Catalytic and reactive polypeptides and methods for their preparation and use

ABSTRACT

Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like. 
     This invention was made with Government support under Grant Contract No. AI-24695, awarded by the Department of health and Human Services, and under Grant Contract No. N 00014-87-K-0256, awarded by the Office of Naval Research. The Government has certain rights in this invention.

This invention was made with Government support under Grant Contract No.AI-24695 awarded by the Department of Health and Human Services, underGrant Contract No. N 00014-87-K-0256, awarded by the Office of NavalResearch. The Government has certain rights in this invention, underGrant Contract CHE 8822412 awarded by the National Science Foundation,and under Grant Subcontract C87-101226 awarded by the Department ofEnergy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 07/404,920, filed Sep. 8,1989 now U.S. Pat. No. 5,215,889, which is a continuation-in-part ofapplication Ser. No. 07/273,455, filed Nov. 18, 1988 (now abandoned).

Application serial number 07/273,786 filed on Nov. 18, 1988, andapplication serial number 07/337,601, filed on Apr. 13, 1989, both ofwhich name Peter Schultz as the sole inventor, contain related subjectmatter. The entire disclosures of these related applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and compositionsproducing novel polypeptides including antibodies capable of promotingchemical reactions. More particularly, the present invention relates tomethods for modifying polypeptides to include active functionalitiescapable of promoting chemical reactions.

The ability to catalyze chemical reactions, such as the synthesis,modification, or cleavage of structurally complex molecules, includingproteins, nucleic acids, carbohydrates, and the like, would be of greatcommercial and scientific benefit. To this end, antibodies have beenprepared having catalytic activity resulting from the ability of theantibody combining site to selectively stabilize transition stateintermediates and to overcome entropic barriers in orienting reactantsin particular reactions.

Although significant catalytic activity has been observed with suchantibodies, it would be desirable to provide catalytic antibodies andother polypeptides having enhanced catalytic activity and specificity.In particular, it would be desirable to be able to design catalyticantibodies and polypeptides having a combining site with a desiredspecificity and affinity for the reactant(s) of interest, whichantibodies and polypeptides further provide catalytic or other reactivegroups proximate the binding site. Such catalytic and reactive groupswould be able to chemically participate in the reaction of interest.

2. Description of the Relevant Art

The preparation of catalytic antibodies against haptens that aretransition state analogs is described in the following references:Pollack et al. (1986) Science 234:1570-1573; Pollack and Schultz (1987)Cold Spring Harbor Symp. Quant. Biol. 52:97-104; Jacobs et al. (1987) J.Am. Chem. Soc. 109:2174-2176; Tramontano et al. (1986) Science234:1566-1570; Tramontano et al. (1988) J. Am. Chem. Soc. 110:2282-2286;and Janda et al. (1988) Science 241:1188-1191. The use of antibodies toovercome entropic barriers involved in orienting reaction partners isdescribed in the following references: Napper et al. (1987) Science237:1041-1043; Jackson et al. (1988) J. Am. Chem. Soc. 110:4841-4842;Janda et al. (1988) J. Am. Chem. Soc. 110:4835-4837; Hilvert et al.(1988) Proc. Natl. Acad. Sci. USA 85:4953-4955; and Berkovic et al.(1988) Proc. Natl. Acad. Sci. USA 85:5355-5358. Polyclonal antibodieshave been generated with cofactor binding sites (Raso and Stollar (1975) Biochemistry 14:584-591). The generation of monoclonal antibodieshaving a cofactor binding site is described in Shokat et al. (1988)Angew. Chem. 100:1227-1229. The generation of affinity labels forantibody combining sites is described by Kohen et al. (1980) FEBS Lett.111:427-431; Metzger et al. (1970) Biochemistry 9:1267-1278; and Givolet al. (1971) Biochemistry 10:3461-3466. Chemically derivatizedhydrophobic and hydrophilic model systems have been shown to afford rateenhancements in hydrolytic and redox reactions by specific substratebinding (Bender et al. (1978) Cyclodextrin Chemistry, Springer-Verlag,Berlin; Tabushi (1982) Acct. Chem. Res. 15:66; Breslow (1982) Science218:532; and Cram et al. (1978) J. Am. Chem. Soc. 106:4987). Both Cramet al. (1976) J. Am. Chem. Soc. 98:1015 and Lehn and Sirlin (1978)J.C.S. Chem. Comm. 949 have demonstrated that polyether macrocyclesderivatized with thiol residues complex and accelerate acyl transfers byfactors of 10³ to 10⁴ in thiolysis reactions of amino acid ester salts(relative to noncomplexing thiols). Furthermore, cyclodextrinsderivatized with pyridoxamines have afforded 100-fold rate accelerationsin transamination of pyruvic acid as well as greater than twenty-foldstereo-selectively in a-amino acid synthesis (Tabushi et al. (1985) J.Am. Chem. Soc. 107:5545; Zimmerman et al. (1983) J. Am. Chem. Soc.105:1694; Breslow et al. (1983) J. Am. Chem. Soc. 105:1390; and Breslowet al. (1980) J. Am. Chem. Soc. 102:423). Baldwin et al. (1975) J. Am.Chem. Soc. 97:227, Collman (1977) Acct. Chem. Res. 10:265; and Traylor(1973) Proc. Natl. Acad. Sci. 78:2647 have shown that cavity containingporphyrins are also capable of mimicing the oxygen binding function ofmyoglobin as well as the oxidative chemistry of P-450 monoxygenase.Introduction of a free thiol into chiral macrocyclic ether has beenshown to promote the transacylation of nitrophenyl glygly ester relativeto uncomplexed dipeptide. Lehn and Sirlin (1978) supra. Site-directedmutagenesis has been used in conjunction with high resolution x-raycrystallography to analyze the structure of enzyme binding sites and thefunction of such sites in catalysis. Wilkinson et al. (1984) Nature307:187-188; Craik et al. (1985) Science 28:291-297; Schultz et al.(1985) Biochemistry 24:6840-6848; Dalbadie-McFarland (1982) Proc. Natl.Acad. Sci. USA 79:6409-6413; and Sigal et al. (1984) J. Biol. Chem.259:5327-5332. A cysteine in the active region of the enzyme papain hasbeen modified with a flavin cofactor. Kaiser et al. (1984) Science226:505-510. A thiol has been introduced into the enzymes staphylococcalnuclease and RNase S and subsequently derivatized with anoligonucleotide. Corey et al. (1987) Science 238:1401-1403. The activesite serine of subtilisin has been chemically converted to a cysteine.Bender et al. (1966) J. Am. Chem. Soc. 88:3153-3154 and Koshland et al.(1966) Proc. Natl. Acad. Sci. USA 56:1606-1611. Antibodies generatedagainst positively charged haptens contain complementary aspartate andglutamate residues (Nisonoff et al. (1975) The Antibody Molecule,Academic Press, pp. 23-27). The experimental data in Example 4 waspublished in Cochran et al. (1988) J. Am. Chem. Soc. 110:7888-7889.

SUMMARY OF THE INVENTION

Using the compositions and methods of the present invention, novelcatalysts and promoters are provided to enhance the rate of chemicalreactions. In some cases, reactions may be catalyzed where no naturalcatalysts exist. The compositions and methods of the present inventionare particularly suitable for the synthesis, modification, and cleavageof structurally complex molecules such as proteins, nucleic acids,carbohydrates, and the like.

According to the present invention, polypeptides are provided which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides include abinding site specific for at least one reactant or reaction intermediateand an active functionality proximate the binding site whichfunctionality is capable of chemically modifying the bound reactant. Theactive functionality may be provided by the side chain of an amino acidlocated proximate the binding site, where the side chain may benaturally-occurring or synthetic (other than naturally-occurring), ormay be provided by a separate catalytic or reactive group covalentlyattached to an amino acid side chain. When the active functionality is anaturally-occurring amino acid side chain or a separate catalytic orreactive functionality covalently attached to a naturally-occurringamino acid side chain, the polypeptide will be other than an enzyme,usually having the structure of an antibody or antibody fragment. Whenthe active functionality is a synthetic amino acid side chain, thepolypeptide may be any polypeptide having the desired binding site,usually having the structure of an antibody, antibody fragment, enzyme,or enzyme fragment. Suitable catalytic and reactive functionalitiesinclude enzyme cofactors, metal complexes, electrophiles, nucleophiles,acidic groups, basic groups, photosynthesizers, alkylating agents,oxidizing agents, and reducing agents.

Methods for preparing catalytic and reactive polypeptides according tothe present invention include both synthetic and recombinant productionof polypeptides where a naturally-occurring amino acid sequence (e.g.,an antibody or enzyme) is modified by the substitution and/or additionof natural and synthetic amino acids proximate the binding site, as wellas post-translational modification of such polypeptides.Post-translational modification of the polypeptides may be accomplishedby the covalent attachment of the active functionality to a suitableside chain of an amino acid proximate the binding site. In some cases,it will be desirable to prepare the polypeptides having a suitable aminoacid for covalent linkage by recombinant techniques, typically byproviding a cysteine, histidine, lysine, serine, tryptophan, ortyrosine, proximate the binding site. Alternatively, polypeptides whichhave not been sequenced may be modified by combining a ligand includingthe active functionality with the polypeptide, where the activefunctionality is cleavably attached to the polypeptide. After the ligandcombines with the binding site, the cleavable active functionality iscovalently attached to an amino acid proximate the binding site. Theattachment of the active functionality to the ligand is then cleaved andthe ligand removed from the polypeptide, leaving the activefunctionality covalently attached proximate the binding site. The activefunctionality may itself comprise the catalytic or reactive group ofinterest, or may be further selectively modified with anotherfunctionality which can act as the catalytic or reactive group ofinterest. In addition to all of these methods, catalytic and reactiveantibodies may be prepared by eliciting them against a hapten having aparticular structure which is chosen to yield antibodies havingpreselected amino acids proximate the binding site. Such antibodies willbe able to bind reactants, reactive intermediates, or transition stateanalogs involved in the reaction of interest, and will further havespecifically located amino acid side chains selected to enhance thereaction rate.

The catalytic and reactive polypeptides of the present invention areuseful for promoting chemical reactions involving the conversion of oneor more reactants to one or more products. A reaction mixture includingthe reactant(s) is exposed to the polypeptide, resulting in promotion ofthe reaction rate. In the case of catalytic functionalities, thepolypeptide will be conserved, while in the case of reactivefunctionalities, the polypeptides will be consumed.

In the specific embodiments, the catalytic and reactive polypeptides maybe prepared by first producing antibodies to a reactant or reactiveintermediate analog, typically a transition state analog ormultisubstrate analog, involved in the chemical reaction of interest.Conveniently, monoclonal antibody techniques will be utilized in orderto obtain a source of homogeneous antibodies of uniform specificity.Once the antibodies are obtained, they will frequently display a certainlevel of catalytic activity based on their ability to stabilize atransition state or strain a ground state involved in a chemicalreaction or their ability to reduce entropic barriers involved inpositioning two or more reactants involved in the reaction. The presentinvention provides for the enhancement of such catalytic activity bymodification of the antibody or a fragment thereof to provide an activefunctionality proximate the binding site defined by the antibody. Theactive functionality may be a naturally-occurring amino acid side chainwhich is positioned relative to the binding site so that it is able tointeract with the reactant(s) or reaction intermediate in such a way topromote the reaction rate. Alternatively, the active functionality maybe a synthetic amino acid side chain or be covalently attached to theside chain of an amino acid located in the proper position relative tothe antibody binding site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the cleavable affinity labels used in Example 1 ofthe Experimental section.

FIG. 2 outlines the methods for synthesizing the affinity labels of FIG.1.

FIG. 3 illustrates the strategy for introducing a thiol functionalityinto the binding site of MOPC 315 in Example 1.

FIG. 4 is a high pressure liquid chromatography profile of the trypticdigest of the heavy chain of Fab labeled with 2 (FIG. 1) monitored by UVabsorbents (solid line) and radioactivity (dashed line), as described inExample 1.

FIG. 5 illustrates the DNP-containing esters utilized as substrates forreaction with thiolated Fab in Example 1.

FIG. 6 is an Eadie-Hofstee plot of cleavage of ester 14 by thiolated Fablabeled with 2 in Example 1.

FIG. 7 illustrates the results of a fluorescence quenching binding assayfor DNP-glycine with fluorescein-Fab adduct (□) versus control withfluorescein plus underivatized Fab ( ). Fluorescence quenchingexperiments were carried out at 10° C. using 492 nm for excitation andmeasuring emission at 521 nm. The fluorescein-Fab adduct was dilutedwith assay buffer (above) to 0.10 μM. Aliquots of 2,4-DNP-glycine wereadded and, after mixing, the fluorescence observed. FreeN-fluoresceinthioureido-2-mercapthoethylamine and underivatized Fab,each at 0.10 μM, were treated in a similar experiment.

FIG. 8 illustrates the strategy for modifying the Fab fragment of MOPC315 to introduce an imidazole at the binding site in Example 2.

FIG. 9 illustrates the hydrolysis reaction catalyzed by theimidazole-antibody adduct of Example 2.

FIG. 10 illustrates the cis, syn-thymine dimer utilized as a hapten inExample 3.

FIG. 11(a) illustrates the increase in absorbants at 272 nm (thyminemonomer) as a function of irradiation time for reaction mixturescontaining 25 μM dimer 1 and 3 μM antibody 15F1-3B1 [ ], 3 μM MOPC 315(dinitrophenyl specific) [•], or no antibody [ ]; and (b) Eadie-Hofsteeplot for the antibody-sensitized cleavage dimer 1.

FIG. 12(a) is an action spectrum showing the dependence of the quantumyield for monomer formation on the wavelength of irradiation. Thereaction mixtures containing 264 μM hapten 2 and 3 μM dimer-specificantibody 15F1-3B1 [Δ] or one of the non-specific antibodies MOPC 315 [□]and MOPC 167 [°]; and (b) fluorescence (arbitrary units:excitation:280nm, emission:348 nm) of the antibody 15F1-3B1 as a function of addedligand 2. Fluorescence quenching experiments were performed with 0.2 μMantibody at 18° C. in the same buffer used for the photolysisexperiments.

FIG. 13 illustrates the elimination reaction of HF from fluoroketonesubstrate 1 and hapten 2 of Example 4.

FIG. 14 (A, B and C) outline the preparation of the site-directedcysteine mutant of antibody S107 in Example 5.

FIG. 15 illustrates the reagents and substrates 1-5 utilized in Example5. In this Figure, "M²⁺ " represents a metal ion with a charge of +2,"PPh₂ " represents diphenyl phosphine, and "X" represents anon-interfering ligand bound to the rhodium atom to complete the valencefor rhodium.

FIG. 16 illustrates the structures of the ligands, substrates, andaffinity labels employed in Example 6 in the Experimental sectionherein.

FIG. 17 illustrates the nucleotide and corresponding amino acidsequences for the synthetic MOPC315 V_(L) described in Example 6 in theExperimental section herein.

FIG. 18 illustrates the expression vector pLcIIFXVL which was used toproduce Y34FcII-V_(L) fusion protein in Example 6 in the Experimentalsection herein.

FIG. 19 is an SDS-PAGE analysis of various stages in the production ofthe mutant Fv proteins produced in Example 6 in the Experimental sectionherein.

FIG. 20 is a gel filtration analysis of Fv(Y34H_(L)) produced in Example6 in the Experimental section herein.

FIG. 21 is a plot of Fv fluorescence titration with DNP-L-lysine asdescribed in Example 6 of the Experimental section herein.Fv(315)(•),Fv(Y34F_(L))(Δ), or Fv(Y34H_(L))(o)(400 μl, 450-500 nM,ε_(280nm) =37,500 M⁻¹) in 100 mM potassium phosphate, pH 6.8 (buffer A),was titrated with solutions of 10-100 μM DNP-L-lysine (ε_(360nm) =17,500M⁻¹) in buffer A while monitoring the protein fluorescence (excitation:282 nm; emission: 343nm). Fluorescence was corrected for the change involume. Dissociation constants (K_(D)) were determined by a modifiedScatchard analysis.

FIG. 22 is a Lineweaver-Burke plot of initial rate data obtained for thehydrolysis of 1 as described in Example 6 of the Experimental sectionherein. Four μl of a 100-fold concentrated solution of substrate 1 inDMSO was added to the 400 μl of Fv (115 nM) in buffer A while monitoring7-hydroxycoumarin fluorescence at 25° C. (excitation:355 nm; emission:457 nm). A standard of 7-hydroxycoumarin was added at the completion ofeach assay to assess quenching of coumarin fluorescence by DNP at highsubstrate concentrations. The buffer background rate was subtracted fromthe initial rates. Each point represents the average velocity of atleast duplicate experiments. Factor X, Factor Xa, or Russell's vipervenom had no hydrolytic activity towards 1.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel catalytic and reactive polypeptides are capable of promoting achemical reaction involving the conversion of reactants to products. Thepolypeptides comprise a binding site capable of specifically attachingto the reactants or reactive intermediates and an active functionalitylocated proximate the binding site. The active functionality willinteract with the reactants or reactive intermediates after binding bythe polypeptide in order to enhance the reaction rate, either by acatalytic or a reactive mechanism. The polypeptides are used bycombining with reactants under conditions suitable for the desiredreaction.

The polypeptides of the present invention may be natural or synthetic,with the term natural generally referring to polypeptides produced byand/or isolated from living biological organisms, e.g., cell culture,animal tissue, plant sources, and the like, and the term syntheticgenerally referring to polypeptides produced by recombinant DNA methodsand by chemical synthesis techniques, e.g., solid phase synthesistechniques. The polypeptides may be a single chain, or include multiplechains, and may be glycosylated or free from glycosylation. The size ofthe polypeptide is not critical, with polypeptides being in the rangefrom 10 kilodaltons (kD) to 1000 kD, usually being in the range from 20kD to 200 kD. The polypeptides may be in the form of natural biologicalproteins, such as antibodies, enzymes, hormones, lectins, cellularreceptors, and fragments thereof, as well as other proteins capable ofspecifically binding target ligands which include reactants, reactiveintermediates, and transition state analogs involved in the chemicalreaction of interest. Usually, in the case of such known forms ofbiologic proteins, the structure of the protein will be modified in apredetermined matter in order to introduce the active functionality ofinterest. In some cases, however, it will be possible to merely alterthe amino acid sequence of the known structure in order to obtain properplacement of the active functionality of interest. Such modifiedproteins will generally retain the ability to mimic thenaturally-occurring protein from which they have been derived and, inparticular, will retain the ability to bind to the target ligand ofinterest.

Alternatively, the polypeptides of the present invention may assumenovel configurations which are not characteristic of known classes ofbiological proteins. In such cases, the polypeptides will besynthesized, either by recombinant techniques or by solid phasesynthesis techniques, to provide a product having a desired sequence andstructural configuration which will allow for binding of the polypeptideto the reactant, reactive intermediate, or transition state analog(target ligand) of interest.

Regardless of form, it is necessary that the polypeptide have a bindingsite with affinity for at least one reactant or reactive intermediate,or transition state analog of the chemical reaction of interest. Theaffinity will be at least about 10⁻³ M⁻¹, usually being at least about10⁻⁴ M⁻¹, preferably being at least about 10⁻⁵ M⁻¹, and more preferablybeing at least about 10⁻⁶ M⁻¹ or higher. In the case of modifiedpolypeptides, i.e., those which have changes in their amino acidsequence or active functionalities covalently bound proximate thebinding site, there should be no substantial loss of binding affinity asa result of the modification, and the modified polypeptide shouldgenerally meet the affinity levels just set forth even if some loss ofaffinity has occurred.

Polypeptides having the desired affinity are most easily prepared byraising antibodies against a reactant, reactive intermediate, ortransition state analog which is involved in the chemical reaction ofinterest. Conveniently, the reactant, reactive intermediate, ortransition state analog is prepared and utilized as a hapten inpreparing the antibodies having desired affinities. Methods forpreparing antibodies to particular transition states are taught inPollack and Schultz (1987) Cold Spring Harbor Symp. Quant. Biol.52:97-104; Jacobs et al. (1987) J. Am. Chem. Soc. 109:2174-2176;Tramontano et al. (1986) Science 234:1566-1570; Napper et al. (1987)Science 237:1041-1043; Jackson et al. (1988) J. Am. Chem. Soc.110:4841-4842; and Janda et al. (1988) J. Am. Chem. Soc. 110:4835-4837,the disclosures of which are incorporated herein by reference.

The binding site of the polypeptide is defined by a grouping of aminoacids which are capable of specifically attaching to the reactant orreactive intermediate of interest. The binding site will usually includefrom about 2 to 15 amino acids, more usually including from about 2 to10 amino acids, where the amino acids may be contiguous or separatedalong the primary sequence of the polypeptide. When the amino acids arenot contiguous, they will usually be brought together by the folding(secondary structure) of the polypeptide. The binding site will usuallydefine a cavity or other three-dimensional structure which iscomplementary with a portion of the reactant or reactive intermediatewhich is being bound. In particular, the side chains of the amino acidswithin the binding site will interact with the particular structure(referred to as an epitope or determinant site in the case of antibodybinding) on the reactant or reactive intermediate being bound.

An active functionality according to the present invention is a moietyor functional group which is covalently attached to the polypeptideproximate the binding site and which is capable of catalytically orreactively interacting with the bound reactant, reactive intermediate,or transition state analog to promote the reaction. The activefunctionality may be located within the three-dimensional structure ofthe binding site or may be located about the periphery of the bindingsite, but will be sufficiently close to the binding site to provide thenecessary interaction with the reactant or reactive intermediate. Theprecise distance between the active functionality and the binding siteis not critical, but will usually be less than about 20 Å, more usuallybeing less than about 10 Å, and preferably being less than about 5 Å.The distance of the active functionality from the binding site, however,may be larger and it is important only that the active functionality onthe polypeptide be sufficiently close to the bound reactant or reactiveintermediate to provide the desired interaction and promotion of thereaction rate.

The polypeptides of the present invention will usually be able topromote a chemical reaction by two distinct mechanisms. By the firstmechanism, the polypeptide binds to a transition state involved in thechemical reaction, stabilizing the transition state and therebyenhancing the reaction rate. By the second mechanism, the activefunctionality will provide a catalytic or reactive interaction with thebound reactant or reactive intermediate. Such interactions will furtherenhance or promote the reaction rate beyond the enhancement attributableto transition state stabilization alone. In some cases, however, thepolypeptides of the present invention will provide only the second ofthese mechanisms.

The active functionalities of the present invention will be capable ofcatalytic interactions as well as reactive interactions where thefunctionality is consumed during the reaction. Such interactions includeproton and hydrogen abstraction, Lewis acid catalysis, electrontransfer, photosensitization, nucleophilic or electrophilic addition tosubstrate, general acid or base catalysis, specific or base catalysis,and the like. Catalytic or reactive groups include naturally-occurringcofactors and derivatives thereof, metal complexes (e.g., chelates),electrophiles, nucleophiles, basic and acidic groups (including Lewisacids), redox active molecules, photoactive molecules, alkylatingagents, reactive radicals, and the like.

The active functionalities of the present invention include virtuallyany chemical group which may be attached to the polypeptide and whichmay be able to provide a desired catalytic or reactive interaction topromote or enhance chemical reaction rate. Generally, such functionalgroups will have a molecular weight in the range from about 80 to 1000daltons (D), usually being in the range from about 100 to 500 D. Theactive functionalities may comprise the side chains of the twentynaturally-occurring amino acids, the side chains of synthetic aminoacids which may be incorporated in the polypeptides by recombinant DNAor other synthetic techniques as described hereinbelow, or a virtuallyunlimited variety of other functional groups which may be attachedthrough the amino acid side chains by conventional protein linkingtechniques.

The naturally-occurring amino acid side chains include the distinctfunctionalities which are found in most biologic systems. Preferably,the amino acid side chains will be selected from the group ofnon-aliphatic side chains, more preferably being selected from the basicside chains (those of lysine, arginine, and histidine), the acidic sidechains (aspartate and glutamate), the aliphatic hydroxyl side chains(serine and threonine), thiol (cysteine), sulfide (methionine), phenol(tyrosine) and aminoindole (trytophan). When employing the natural aminoacid side chains as the active functionalities of the present invention,it will be necessary to arrange the side chains so that theyspecifically interact with the reactant or reactive intermediate in amanner which enhances the reaction rate beyond that which isattributable to any transition state stabilization which occurs.Usually, when the active functionalities are the amino acid side chains,they will be part of the amino acids which define the binding site,although it is possible for the amino acids outside of the binding siteto assume a catalytic or reactive activity.

In addition to the natural amino acid side chains, synthetic amino acidshaving non-natural side chains (or other variations, e.g., D-isomers)may be incorporated in the polypeptide structure, either by recombinantDNA techniques or by solid phase synthesis techniques, as described inmore detail hereinbelow. Such synthetic amino acids may have a widevariety of side chains which are not normally found inbiologically-produced proteins, providing an array of reactive andcatalytic capabilities which could not be found in natural enzymes.

Synthetic amino acid side chains which may be incorporated in thepolypeptides of the present invention (either by employing syntheticamino acids or by covalent attachment to natural or synthetic amino acidside chains as described below) include both catalytic functionalities,such as acids, bases, enzyme cofactors, metal complexes, electrophiles,nucleophiles, photoactive molecules, redox active molecules, and thelike, and reactive funtionalities, such as alkylating agents, oxidizingagents, reducing agents, hydrolytic agents, photoactive agents, and thelike. Specific functionalities include thiols, primary and secondaryamines, aldehydes, carboxylates, nitriles, aromatic amines, aromaticcarboxylates, primary alcohols, and the like.

Active functionalities may also be covalently attached to the sidechains of amino acids proximate the binding site. The choice of suchcovalently-attached active functionalities is virtually unlimited,although functionalities within the binding site will usually besufficiently small so as not to interfere with the desired bindingability of the polypeptide. Often, the active functionalities must becapable of covalent attachment to amino acid side chains by suitablechemical synthesis techniques under conditions which do not denature thepolypeptide.

Suitable active functionalities which may be covalently attached to thepolypeptides of the present invention by binding to amino acid sidechains include naturally-occurring cofactors and derivatives thereof,such as pyridoxamine (useful in transamination, racemization, ordecarboxylation reactions), flavins and nicotinamides (useful in redoxreactions), porphyrins (useful in hydroxylation or epoxidationreactions), thiamine (useful in decarboxylation and addition reactions),and folates (useful in redox reactions). Synthetic activefunctionalities include metal complexes, such as Zn⁺² -phenanthrolinederivatives (useful in hydrolytic and redox reactions), Fe^(III) -EDTA(ethylenediaminetetraacetic acid) derivatives (useful in redox reactionsincluding hydroxylations), Co^(II) and Co^(III) -polyamine derivatives(useful in hydrolytic reactions), Cu⁺² -bipyridyl derivatives (useful inredox reactions including hydroxylations), Rh^(II) -phosphine and aminederivatives (useful in hydrogenation and hydrolytic reactions) and Zn⁺²cyclam derivatives; photosensitizers, such as indoles, quinones, andbenzophenones (useful in photocleavage, photoaddition,photorearrangement, photoisomerization, and singlet oxygen formation);nucleophiles, such as hydroxamates, hydroxylamines; hydrazines, andperoxides (useful in hydrolytic reactions); bases, such as pyridines,tetrazoles, alkylamines (useful in elimination, hydrolytic, andisomerization reactions); electrophiles, such as nitrogen and sulfurmustards, imines, aldehydes, alkyl halides, sulfates, sulfonates, andquinones (useful in addition reactions); and the like.

The distinction between active functionalities having catalytic activityand reactive activity is as follows. Catalytically activefunctionalities lower the free energy of activation for a reactionthereby accelerating the rate of reaction. The catalytically activefunctionality itself undergoes no net chemical or structural change atthe completion of the reaction. Reactive functionalities, in contrast,cause a chemical transformation in one or more of the reactants andthemselves will undergo a net chemical change at the completion of thereaction.

The catalytic and reactive polypeptides of the present invention may beprepared by a number of alternate techniques. The techniques includeboth synthetic and recombinant preparation methods where the polypeptideis produced to have a specific amino acid sequence (with the activefunctionality usually being a natural or synthetic amino acid sidechain) as well as post-translational modification methods where theactive functionality is covalently attached to an amino acid side chainof a previously-prepared polypeptide by chemical linking reactions. Insome cases, however, it will be desirable to combine the two approachesby first producing a polypeptide having a preselected natural orsynthetic amino acid located at a desired position and then covalentlyattaching an active functionality to the side chain of the preselectedamino acid by suitable post-translational chemical linking methods.

A third general approach involves the preparation of antibodies having adesired binding site configuration based on the geometry and electronicconfiguration of the hapten used to raise the antibody. In addition tomimicing the reactant or reactive intermediate, the hapten will bechosen to elicit the desired catalytic or reactive amino acid side chainwithin the antibody binding site. Specifically, the hapten may be areactant, reactant analog, reactive intermediate, reactive intermediateanalog, transition state analog, or multisubstrate analog which furtherpossess structural and electronic features which generate at least onepreselected amino acid proximate the antibody combining site. The sidechain of that amino acid will be the active functionality, as definedabove, in the resulting antibody molecules.

Synthetic and recombinant techniques for producing the catalytic andreactive polypeptides of the present invention will usually start withan exemplary amino acid sequence which is characteristic of a proteinwhich is known to bind a reactant or reactive intermediate with therequisite affinity. Such exemplary amino acid sequences may be derivedfrom enzymes which are known to catalyze the chemical reaction ofinterest or from antibodies which have been raised against reactants ortransition state and multisubstrate analogs of the reaction of interest.The preparation of such antibodies is well described in the backgroundart described above. In addition to the desired binding affinity, theexemplary protein may also possess catalytic activity as will frequentlybe the case with enzymes and antibodies which have been elicited againsta hapten which mimics a transition state analog. In such cases, thepresent invention may be utilized to enhance the catalytic or reactivecapability of the modified polypeptides relative to the exemplaryproteins from which they are derived.

Once the amino acid sequence of the exemplary protein is obtained, itwill be possible to examine the interaction between the protein and thereactant or reaction intermediate and determine the desiredmodification, i.e., locate where to introduce an active functionality.Conveniently, x-ray crystallography or other suitable analytictechniques may be used to study the orientations and interactions of thereactants or reactive intermediates with the exemplary protein in orderto determine what modification would be beneficial.

In the case of amino acid substitutions or additions which will bemodified post-translationally with catalytic or reactive groups, thedesired modification will be made so as to place the catalytic orreactive group in close proximity to the bound substrate. If possible,unfavorable steric interactions between the catalytic or reactive groupand the substrate will be minimized. In addition, orbital overlapbetween the catalytic or reactive group and the substrate will beoptimized. The modification should also be made so as to facilitatepost-translational modification of the amino acid.

In the case of substitution or addition of catalytic or reactive naturalor synthetic amino acid side chains, modification will be made so as tominimize unfavorable steric interactions between the polypeptide andsubstrate so as to optimize orbital overlap between the side chain andthe substrate. In all cases, it is important that the modification doesnot significantly decrease the binding affinity of the polypeptide tothe substrate or significantly destabilize the polypeptide itself.

Synthetic methods may be utilized for preparing polypeptides havingfewer than about 200 amino acids, usually having fewer than about 150amino acids, and more usually having 100 or fewer amino acids. Thesynthetic preparation methods, as described in more detail below, areparticularly convenient for allowing the insertion of synthetic(non-natural) amino acids at a desired location within the polypeptide,but suffer from low yields and lack of glycosylation when compared tobiological synthesis techniques.

Suitable synthetic polypeptide preparation methods may be based on thewell-known Merrifield solid-phase synthesis method where amino acids aresequentially added to a growing chain (Merrifield (1963) J. Am. Chem.Soc. 85:2149-2156). Automated systems for synthesizing polypeptides bysuch techniques are now commercially available from suppliers such asApplied Biosystems, Inc., Foster City, Calif. 94404; New BrunswickScientific, Edison, N.J. 08818; and Pharmacia, Inc., BiotechnologyGroup, Piscataway, N.J. 08854.

For the preparation of larger and/or glycosylated polypeptides,recombinant preparation techniques are usually preferred. Suchrecombinant techniques involve the expression in cultured cells ofrecombinant DNA molecules encoding the desired polypeptide amino acidsequence. The DNA sequence may itself be synthetic or alternatively bemodified from a natural source, i.e., the gene of an exemplary antibodyor enzyme. Synthetic DNA sequences (polynucleotides) may be synthesizedby well-known techniques. For example, short-single stranded DNAfragments may be prepared by the phosphoramidite method described byBeaucage and Carruthers (1981) Tett. Letters 22:1859-1862. Adouble-stranded fragment may then be obtained by either synthesizing acomplementary strand and annealing the strands together underappropriate conditions or by adding the complementary strand using DNApolymerase with an appropriate primer sequence. Conveniently, automatedequipment for preparing the synthetic DNA sequences are available fromthe suppliers listed above as providing synthetic polypeptide equipment.Alternatively, the desired DNA sequence may be obtained from a suitablecDNA or genomic library obtained from a cell line expressing theexemplary protein of interest. For example, the gene expressing amonoclonal antibody of interest may be isolated from the hybridoma cellline expressing such antibody. The gene may then be modified asdescribed in more detail hereinbelow to substitute or add the aminoacids providing the active functionality (or active functionalityattachment site) of interest. The techniques for isolating antibodygenes from hybridoma cell lines are well described in the scientificliterature. See, for example, Gearhart et al. (1983) Proc. Natl. Acad.Sci. USA 80:3439-3443.

The natural or synthetic DNA fragments coding for the desired catalyticor reactive polypeptide will be incorporated in DNA constructs capableof introduction to and expression in an in vitro cell culture. The DNAconstructs may be suitable for replication in a unicellular host, suchas yeast or bacteria, but will frequently be intended for introductioninto and integration within the genome of cultured mammalian or othereukaryotic cell lines. DNA constructs prepared for introduction intobacteria or yeast will include a replication system recognized by thehost, the DNA fragment encoding the polypeptide of interest,transcriptional and translation initiation regulatory sequences joinedto the 5'-end of the DNA sequence, and transcriptional and translationaltermination regulatory sequences joined at the 3'-end of the DNAsequence. The transcriptional regulatory sequences will include aheterologous promoter which is recognized by the host. Conveniently,available expression vectors which include replication system andtranscriptional and translational regulatory sequences together with aninsertion site for the DNA sequence to be expressed may be employed.

Of particular interest to the present invention are expression systemsfor the Fv and F(ab) regions of an antibody molecule. Such systems aredescribed in Bird et al. (1988) Science 242:423; Houston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879; Skerra and Pluckthun (1988) Science240:1028; and Bette et al. (1988) Science 240:1041. Polypeptidescomprising these regions retain the binding specificity of the intactantibody from which they are derived, but are substantially smaller andmay be produced in a variety of expression hosts, e.g., E. coli, whichoffers advantages over the production of intact monoclonal antibodies inhybridoma cell lines.

It will frequently be desirable to produce both the V_(L) and V_(H)chains of the Fv region as a single fusion protein joined by anappropriate linker which allows folding. Such single chain expressionsystems are described in Bird et al. (1988), supra, and Houston et al.(1988), supra. Alternatively, the V_(L) and V_(H) chains may beexpressed and subsequently reconstituted under appropriate conditions.Such separate chain expression systems are described in Skerra andPluckthun (1988), supra, and Bette et al. (1988) supra.

When starting with isolated genes encoding the exemplary protein,usually an antibody, enzyme, or a fragment thereof, it is necessary tomodify the gene to substitute or add amino acids within or near thebinding site. A variety of methods for altering the natural genesequence to provide such substitutions or additions are known and amplydescribed in the patent and scientific literature. See, for example,Molecular Cloning: A Laboratory Manual (Maniatis et al., eds.) ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) and Guide toMolecular Cloning Techniques (Berger and Kimmel, eds.) Methods inEnzymology, Vol. 152, Academic Press, Inc., Orlando, Fla. (1987).Particular techniques for providing such alterations in the gene includesite-directed mutagenesis as described by Kunkel et al. (1985) NucleicAcids Res. 13:8764 and cassette mutagenesis as described by Wells et al.(1985) Gene 34:315-323. The altered gene may then be expressed in cellculture as described above and the modified polypeptide obtained byconventional purification techniques.

Site-directed mutagenesis is particularly useful for modifying theprimary structure of antibodies and antibody fragments, where theantibodies or fragments possess binding regions which are capable ofcombining with the reactant(s) or reactive intermediate(s) of interest.Suitable antibody fragments will include at least a portion of one ormore of the complementarity-determining regions (CDR's) of the variableregion of the intact antibody, usually including at least one intactCDR, more usually including at least two intact CDR's, and preferablyincluding all of the CDR's present in the corresponding intact antibody(where the number will vary depending on the species of origin of theintact antibody). Particularly useful will be F(ab) fragments, Fvfragments, V_(H) fragments, and V_(L) fragments.

Once a desired antibody is selected as a model for preparing thepolypeptide of the present invention, the antibody or antibody gene issequenced. Suitable methods for sequencing the amino acids of theantibody itself are described in Hochman et al. (1973) Biochem. 12:1130while suitable methods for isolating and sequencing the antibody genefrom a hybridoma cell line are described in Gearhardt et al. (1983),supra. When only a fragment of the antibody is to be employed, e.g., theF(ab), Fv, V_(L), or V_(H), it will be necessary to sequence only thecorresponding portion of the antibody or antibody gene, althoughfrequently the entire molecule will be sequenced.

Once determined, the amino acid or DNA sequence of the antibody can beused to devise a DNA sequence encoding the desired catalytic or reactivepolypeptide. The DNA sequence will normally utilize the preferred codonsfor the intended expression host and will further incorporate one ormore unique restriction sites to facilitate cloning and subsequentmutagenesis of the synthetic gene.

When devising the DNA sequence, it will be possible to incorporate avariety of modifications intended to impart or enhance the catalytic orreactive activity of the polypeptide which is to be produced. Asdiscussed above, it will be possible to substitute a variety of naturalamino acids having active side chains at preselected locations proximatethe CDR's. Additionally, it will be possible to substitute entire CDR'sor portions thereof with regions derived from other proteins, e.g.,metal binding sequences from metalloproteins, active regions fromenzymes, CDR's from other antibodies, peptide hormone binding sites, andthe like.

Site-directed mutagenesis will also be useful for introducing naturalamino acids having side chains which may be used for covalent attachmentof active functionalities. Particularly useful are cysteines havingthiol side chains which can be covalently attached to thiols present onthe active functionality, typically by disulfide exchange or alkylationreactions.

In addition to site-directed mutagenesis, random mutagenesis techniquesmay be employed to modify the primary structure of the antibodies andantibody fragments of the present invention. Typically, bacterial orother cell lines producing the antibodies will be exposed to knownmutagens, such as sodium bisulfite, and the mutagenized antibodiesscreened or selected for catalytic or reactive activity using standardtechniques. See, e.g., Youderian et al. (1983) Cell 35:777 and Das(1989) Proc. Natl. Acad. Sci. USA 86:496. Antibodies displaying enhancedactivity may then be selected and further tested for suitability ascatalytic or reactive polypeptides according to the present invention.

In some cases, it may be desirable to introduce specific substitutionsusing site-directed mutagenesis as described above, where the effect ofsuch substitution is unpredictable. In those cases, the resultingantibodies and antibody fragments will have to be screened in order todetermine whether there is an observable effect on catalytic activity orreactivity. This approach may be particularly suitable for identifyingregions within the antibody or antibody fragment which have an effect onthe activity. Once the region is identified, further modification can beeffected in order to further enhance the catalytic activity orreactivity.

An alternative method may be employed for the substitution of synthetic(non-natural) amino acids in an exemplary protein encoded by anunmodified protein gene. The gene is altered, asdescribed above, toreplace an amino acid codon at the desired location proximate thebinding site with a nonsense codon differing from the gene's terminationcodon. Conveniently, oligonucleotide-directed mutagenesis can beemployed for such substitution. A suppressor tRNA, directed against thenonsense codon, is chemically or enzymatically acylated with the desiredsynthetic amino acid and added to an in vitro transcription/translationsystem programmed to express the altered DNA sequence. The syntheticamino acid will be inserted at the location of the nonsense codon. Theapproach, of course, would work with natural amino acids as well, butwould be more cumbersome than simple alteration of the DNA sequence toencode the desired natural amino acid.

The desired unique codon must not encode a natural amino acid insertionsite (natural tRNA recognition site), and a suitable codon is a TAGtermination or amber codon. Any mutation in a gene that generates atermination codon (TAG, TAA, or TGA) leads to premature termination ofpolypeptide synthesis as a consequence of the inability of thenaturally-occurring tRNA to bind and compete with the release factors.Such altered genes will, of course, have to utilize one of the twoalternate termination codons. This approach is particularly advantageousin that alternate amino acid substitutions can be obtained bypreparation of different synthetic amino acids at the aminoacylationstage.

Mutations in the anti-codon loop of a number of tRNA molecules leads toan amber suppressor tRNA. See, e.g., Steege and Soll, in: BiologicalRegulation and Development, Vol. I, (Goldberger, ed.) Plenum Press(1978). Amber suppressors, characterized by a 5'-CUA-3' anti-codon loop,no longer respond to codons recognized by their wild-type counterparts,but instead insert amino acids only in response to amber codons (UAG).The desired tRNA carrying a synthetic amino acid can be generated byanti-codon loop replacement, as described by Bruce et al. (1982) Proc.Natl. Acad. Sci. USA 79:7127-7131. Alternatively, a gene encoding thetRNA carrying the synthetic amino acid may be synthesized and expressedin a suitable expression system or the suppressor tRNA can be generatedby runoff transcription. The chemically or genetically constructedsuppressor tRNA must then be aminoacylated with the synthetic amino acidto be incorporated into the polypeptide of the present invention. Asuitable chemical acylation method which utilizes N-blocked aminoacylpCpA dinucleotide synthesized via carbonyl-diimidazole mediated couplingof N-blocked amino acids with protected pCpA-OH, is condensed with anabbreviated tRNA in the presence of T4 RNA ligase. Heckler et al. (1984)Tetrahedron 40:87-94. A suitable prokaryotic in vitrotranscription/translation system is described in Pratt, in:Transcription and Translation, A Practical Approach (Hames and Higgens,eds.) IRL Press, Washington (1984).

The recombinant production of antibodies (immunoglobulins) and theirvarious modifications is taught in a number of recent patentapplications, including EPO 8430268.0; EPO 85102665.8; EPO 85305604.2;PCT/GB85/00392; EPO 85115311.4; PCT/US86/002269; and Japaneseapplication 85239543, the disclosures of which are incorporated hereinby reference. See also U.S. Pat. No. 4,518,584, the disclosure of whichis incorporated herein by reference, which describes site-directedmutagenesis of mammalian proteins.

Post-translational modification of exemplary proteins may be obtained bya novel technique employing cleavable affinity-labeling reagents. Suchreagents are obtained by joining cleavable tethers or cross-links toaffinity labels, e.g., haptens specific for the protein binding site. Bybinding the cleavable affinity label to the protein at the binding site,and subsequently covalently attaching the free end of the tether to anamino acid side chain proximate the binding site, the binding ligand canbe cleaved and removed from the protein, leaving the label including adesired active functionality in place. Useful side chains for attachmentinclude carboxylate (aspartate and glutamate), primary amine (lysine),imidazole (histidine), phenol (tyrosine), and thiol (cysteine). Specificmethods for synthesizing such affinity labels and attaching them to anantibody combining site are taught in Example 1 in the Experimentalsection hereinafter. Such cleavable affinity labels are particularlysuitable for selectively modifying polypeptides which have not beenstructurally characterized.

The introduction of thiols to the binding site of a polypeptide may beaccomplished using affinity labels as follows. The cleavable affinitylabel may be a thiol ester or a disulfide, and cleavage of the labelafter site-specific derivatization will incorporate a free thiol at thebinding site. A thiol ester can be cleaved with hydroxylamine while adisulfide can be reduced with dithiothreitol. The disulfides are reducedunder mild conditions chosen to avoid disulfides buried within theprotein. Another functionality suitable for cleavable linkage is acetal,which would lead to a protein-bound aldehyde that could be derivatizedby reductive amination. However, the acidic conditions required forcleavage could lead to inactivation of acid-sensitive proteins. Anazobenzene crosslink could be cleaved reductively to give an aromaticamine, which could then be selectively derivatized at low pH.

The choice of the reactive functionality in the affinity-labellingreagents is governed by a number of factors. The reactive group shouldreact slowly enough so that it will react primarily at or near thebinding site of the protein after equilibrium is established. The labelmay react with more than one type of amino acid residue, which isadvantageous for maximizing the likelihood of successful derivatization.It is disadvantageous in that it can complicate the characterization ofthe labeled protein. Ideally, a unique residue on the polypeptide shouldbe modified to give a homogeneous adduct. Another importantconsideration is the synthesis of the labeling reagents. It should bepossible to easily vary the length of the reagent in order to optimizeincorporation of label into the protein. The affinity label should alsobe easily synthesized in radiolabeled form, for ease in quantitatinglabel incorporation. Finally, the label should be stable to subsequentprotein manipulation conditions, including the degradative methods usedin protein sequence determination.

Two preferred affinity reactive labeling groups are diazoketones andaromatic azides. These groups can be transformed into reactive speciesin the protein binding site by photochemical irradiation and thereforehave the advantage that they can be specifically activated in thebinding site of a protein. Several other reactive chemicalfunctionalities are also suitable. Diazonium groups react specificallywith the phenolic side chain of tyrosine. Epoxide groups react withnucleophilic side chains including primary amines (lysine) andcarboxylates (aspartate and glutamate). Aldehydes react with the primaryamine side chain of lysine to form Schiff base intermediates which canbe reduced with sodium cyanoborohydride. The use of tritiated NaCNB³ H₃leads to tritium incorporation, facilitating the characterization of thelabeled protein. Finally, a-bromoketones are versatile affinity-labelingreagents. Their reactivity is sufficiently slow that they can labelproteins nearly stoichiometrically in their binding sites. Moreover,they react with a number of nucleophilic side chains, includingcarboxylate, primary amine (lysine), imidazole (histidine), and phenol(tyrosine).

The catalytic and reactive polypeptides of the present invention areutilized to promote the chemical reactions for which they have beenprepared. In general, it is necessary that the polypeptides be exposedto a reaction mixture including the chemical reactants and/or reactiveintermediates in such a manner that the binding sites of thepolypeptides will be exposed to the system. In the case of polypeptideswhich are soluble in the reaction mixture of interest, it may bepossible to simply dissolve a preselected concentration of thepolypeptides in the reaction mixture. In other cases, it may benecessary to immobilize the polypeptides on a solid phase, such as asolid surface, porous beads, gels, and the like, or to dissolve orsuspend the polypeptides in organic solvents, reverse micelles, orbiphasic solvent systems.

According to the present invention, polypeptides with catalytic orreactive groups will have a host of applications. Catalytic polypeptideshaving active functionalities such as metal ligand complexes,nucleophiles, acids, and bases (or a combination thereof) can act ascatalysts for the selective cleavage or formation of amides inpolypeptide bonds (in peptides and proteins), glycosidic bonds (insugars) or phosphodiester bonds (in RNA and DNA). Such catalysts wouldbe useful for synthesizing biologically active polypeptides and sugarsor modifying existing biologically active polypeptides, sugars ornucleic acids so as to increase or diminish their inherent biologicalactivity. For example, a sequence specific peptidase could be generatedwhich would enable us to selectively degrade polypeptides.

Polypeptides having metal ligand complexes, photosensitizers, naturalcofactors, and redox active molecules as active functionalities can actas catalysts for the synthesis or modification of synthetic compounds ornatural products, or for modifying biologically active molecules such aspolypeptides, sugars or nucleic acids so as to increase or diminishtheir inherent biological activity. For example, DNA could beselectively cleaved by oxidation using an antibody having EDTA Fe^(II)as an active functionality.

Incorporation of reactive groups such as alkylating agents (nitrogen orsulfur mustard) into antibodies or other polypeptides could lead to areactive polypeptide capable of irreversibly binding and alkylating areceptor on the surface of an organism.

Active functionalities may be introduced to the polypeptides of thepresent invention by attachment to unique or rare amino acid side chainswhich have been introduced to the polypeptides. For example, it ispossible to introduce unique synthetic amino acid side chains by themethods discussed above. By employing linking chemistry specific forsuch synthetic side chains, additional active functionalities may becoupled to the polypeptide. Alternatively, the introduction of aminoacids having naturally reactive side chains, such as cysteine,histidine, lysine, and tyrosine, may also serve as the basis forcovalent attachment of other functionalities. In some cases, however,care must be taken to prevent attachment of the functionalities to theamino acids located elsewhere in the polypeptide. Thus, it will often bepreferable to utilize relatively rare amino acids, such as cysteine,rather than common amino acids, such as lysine. Suitable chemistries forattaching the functionalities to each of these side chains are welldescribed in the chemical and patent literature.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL Example 1

Introduction of Active Functionality to Antibody Using Affinity Label.

The following studies were carried out on the IgA MOPC315, which bindssubstituted 2,4-dinitrophenyl (DNP) ligands with association constantsranging from 5×10⁴ to 10⁶ M⁻¹ (Haselkorn et al. (1974) Biochemistry13:2210). Although a three-dimensional structure is not available, theantibody combining site has been characterized by spectroscopic methods(UV, fluorometry, NMR), chemical modification, and amino acid sequencingof the variable region. Moreover, earlier affinity-labeling studies withreagents of varying structures (Givol and Wilcheck (1977) Meth. Enzy.46:479; Givol et al. (1971) Biochemistry 10:3461; and Strausbauch et al.(1971) Biochemistry 10:4342) defined a number of reactive amino acidside chains in the vicinity of the combining site.

Cleavable tethers were incorporated into affinity labels specific forthe MOPC315 combining site, as shown in FIG. 1. These affinity-labelingreagents contain the DNP group linked to electrophilic aldehyde ora-bromoketone groups via cleavable disulfide or thiophenyl linkages.Covalent attachment of the label to the antibody, followed by cleavageof the crosslink and removal of the free ligand, results insite-specific incorporation of a free thiol into the antibody combiningsite. The geometry of affinity-labeling reagents 1-5 varies with regardto the distance between the DNP group and electrophilic moiety since theposition of a nucleophilic lysine, histidine, or tyrosine side chain isnot precisely known. The syntheses of affinity labels 1-5 are outlinedin FIG. 2, and are described in detail hereinbelow.

(N-2,4-Dinitrophenyl)-2-aminoethyl 2-(1,3-dioxolanyl)⁻ ethyl disulfide6.

Affinity labels 1 and 2 were prepared as follows:

To a suspension of N,N'-bis-(2,4-dinitrophenyl)-cystamine (Chan et al.(1979) Phosphorous Sulfur 7:41) (4.84 g, 10 millimoles) indimethylformamide (200 ml) with triethylamine (50 μl) was added2-(2-mercaptoethyl)-1,3-dioxolane (2.45 g, 18 millimoles), and themixture was stirred at 60° C. for 24 hours. Hydrogen peroxide (2 ml of a30% solution) was added and the dimethylformamide was removed in vacuo.

The residual oil was dissolved in methylene chloride (100 ml). Themethylene chloride layer was washed with water (3×5 ml), dried overMgSO₄, and concentrated in vacuo to an oil. Silica gel columnchromatography with 3:2 hexanes: ethyl acetate (R_(f) -0.31) afforded 6(1.37 g, 3.65 millimoles, 20%) as an orange oil. IR (thin film): 3353,2923, 1623, 1525, 1342, 1138 cm⁻¹ ; ¹ H-NMR, CDCl₃ : w 2.08 (m,2H),2.82(t,2H,J=7.1 Hz), 3.00 (t,2H,J=6.6 Hz), 3.8-4.0(m,6H), 4.95 (t,1H,J=4.3Hz), 7.00(d, 1H,J=9.5 Hz), 8.30 (d, 1H,J=9.6 Hz), 8.80 (s, 1H), 9.16 (d,1H, J=2.6 Hz); mass spectrum (EI) 375 (M+). Anal. Calcd. for C₁₃ H₁₇ N₃O₆ S₂ : C, 41.60; H, 4.53; N, 11.20; S, 17.07. Found: C, 41.57; H, 4.67;N, 11.10; S, 16.99.

(N-2,4-Dinitrophenyl)-2-aminoethyl 3-oxopropyl disulfide 1.

A solution of dioxolane 6 (0.50 g, 1.33 millimoles) in water (2 ml),acetonitrile (2 ml) and acetic acid (8 ml) was heated at reflux for 20h. At this time the mixture was cooled to room temperature and addedslowly to 150 ml of cold saturated aqueous NaHCO₃. The aqueous layer wasextracted with methylene chloride (3×50 ml). The combined extracts weredried over MgSO₄ and concentrated in vacuo to an orange oil. Silica gelchromatography using a gradient of 30 to 40% ethyl acetate in hexanesafforded 1 (0.30 g, 0.91 millimole, 68%) as an orange semi-solid: R_(f)=0.22 in 3:2 hexanes: ethyl acetate; IR (KBr pellet) 3353, 1722, 1623,1525, 1504, 1426, 1342, 1131 cm⁻¹ ; ¹ H-NMR (CDCl₃) δ 2.90-3.04 (m, 6H),3.79 (t,2H,J=6.5 Hz), 7.01 (d,1H,J=9.5 Hz), 8.31 (d,1H,J=9.5 Hz), 8.80(m,1H), 9.15 (d,1H,J=2.6 Hz) 9.83 (s, 1H); mass spectrum (EI) 331 (M+).Anal. Calcd. for C₁₁ H₁₃ N₃ O₅ S₂ : C,39.88; H, 3.93; N, 12.69; S,19.34. Found: C, 40.01; H, 3.91; N, 12.66; S, 19.34.

(N-2,4-Dinitrophenyl)-2-aminoethyl 3-(1,3-dioxolanyl)-propyl disulfide7.

This compound was prepared as described above for 6, starting withN,N'-bis-(2,4-dinitrophenyl)-cystamine (2.42 g, 5 millimoles) and3-(3-mercaptopropyl)-1,3-dioxolane (1.48 g, 10 millimoles), to give 7(0.60 g, 1.54 millimoles, 15%) as an orange oil: R_(f) =0.43 in 3:2hexanes: ethyl acetate; IR (thin film) 3360, 2923, 1623, 1592, 1528,1426, 1335, 1131: cm⁻¹ ; ¹ H-NMR (CDCl₃); δ 1.80-1.95 (m,6h), 2.78(t,2H,J=7.0 Hz), 2.99 (t, 2H,J=6.7 Hz), 3.8-4.0 (m, 6H), 4.87 (t, 1H,J=4.2 Hz), 7.01 (d, 1H, J=9.5 Hz), 8.32 (d, 1H, J=9.5 Hz), 8.79 (s, 1H),9.16 (d, 1H, J=2.6 Hz); mass spectrum (EI) 389 (M), 368, 359, 294, 279,256, 225, 210, 196. Anal. Calcd. for C₁₄ H₁₉ N₃ O.sub. 6 S₂ : C, 43.19;H, 4.88; N, 10.80; S, 16.45. Found: C, 43.27; H, 4.65; N, 10.79; S,16.40.

(N-2,4 Dinitrophenyl-2-aminoethyl 4-oxobutyl disulfide 2.

This compound was prepared as described above for 1, starting withdioxolane 6 (389 mg, 1.0 millimole), to give 2 (340 mg, 0.99 millimole,99%) as an orange solid: mp 64°-65° C.; R_(f) =0.25 in 3:2 hexanes:ethyl acetate; IR (KBr pellet) 3331, 3092, 2923, 2846, 1722, 1630, 1589,1525, 1415, 1342, 1247, 1146 cm⁻¹ ; ¹ H-NMR (CDCl₃) δ 2.06 (t,2H, J=7.3Hz), 2.63 (t, 2H, J=7.0 Hz), 2.75 (t, 2H, J=7.1 Hz), 3.00 (t, 2H, J=6.6Hz), 3.81 (t, 2H,J 6.1 Hz), 7.01 (d, 1H, J=9.5 Hz), 8.31 (d, 1H, J=9.5Hz), 8.79 (s, 1H), 9.16 (d, 1H, J=2.6 Hz), 9.80 (s, 1H); mass spectrum(EI) 345 (M). Anal. Calcd. for C₁₂ H₁₅ N₃ O₅ S₂ : C, 41.74; H, 4.35; N,12.17; S, 18.55. Found: C, 41.71; H, 4.38; N, 12.11; S, 18.49.

Affinity labels 3, 4, and 5 were prepared as follows:

3-(S-DNP)-mercaptopropanoic acid 8.

To a solution of 3-mercaptopropanoic acid (1.59 g, 15 millimoles) in 70ml of 2.0M aqueous sodium acetate (pH 5.2) was added a solution of2,4-dinitrofluorobenzene (3.32 g, 18.0 millimoles) in 50 ml of absoluteethanol over 15 min with stirring at room temperature under nitrogen.After 24 hours, the pale yellow solid precipitate was collected andwashed with water to give 8 (3.40 g, 12.5 millimoles, 83%): mp154.5°-157.5° C.; IR (KBr) 3630,3476 (br), 3113, 3082, 1718, 1589,1511,1344,1055,922 cm⁻¹ ; ¹ H-NMR (acetone-d₆) δ 2.71 (t, 2H, J=7.5 Hz),3.40 (t, 2H, J=7.5 Hz), 3.40 (t, 2H, J=7.4 Hz), 3.40 (s, 1H), 7.87 (d,1H, J=9.1 Hz), 8.32 (dd, 1H, J=2.5, 9.0 Hz), 8.76 (d, 1H, J=2.5 Hz);mass spectrum (FAB⁻) 271 (M-H)⁻, 249, 199, 183, 141, 113, 109. Anal.Calcd. for C₉ H₈ N₂ O₆ S: C, 39.71; H, 2.96; N, 10.29; S, 11.78. Found:C, 39.92; H, 2.96; N, 10.15; S, 11.64.

To a solution of 8 (0.70 g, 2.5 millimoles) in dry dioxane (10 ml) wasadded thionyl chloride (5 ml). The mixture was stirred under nitrogenfor 12 hours. The volatiles were removed in vacuo and the yellow residuewas dissolved in dry dioxane (25 ml). A solution of diazomethane (0.6M)in diethyl ether (25 ml) was added. The mixture was stirred at 0° C. for4 hours after which the volatiles were removed in vacuo. The residue waspurified by silica gel chromatography using 9:1 methylene chloride:ethyl acetate as the eluent to give 9 (0.29 g, 0.92 millimoles, 36%) asa yellow solid: mp 117°-119° C.; R_(f) =0.52 in above eluent; IR (KBrpellet) 3458 (br), 3090, 2111, 1637, 1583, 1508, 1340, 1096 cm⁻¹ ; ¹H-NMR (CDCl₃) δ 2.75 (t, 2H, J=7.1 HZ), 3.35 (t, 2H, J=7.3 Hz), 5.32 (s,1H), 7.61 (d, 1H,J=9.0 Hz), 8.48 (dd, 1H, J=2.5, 9.0 Hz), 9.07 (d, 1H,J=2.5 Hz); mass spectrum (FAB⁻) 296(M⁻), 199, 183, 141, 109. Anal.Calcd. for C₁₀ H₈ N₄ O₅ S: C, 40.54; H, 2.72; N, 18.91; S, 10.82. Found:C, 40.81, H, 2.89; N, 18.59; S, 10.79.

1-Bromo-2-oxo-4-(S-DNP)-mercaptobutane 3.

To a solution of 9 (95 mg, 0.32 millimoles) in dry dioxane (5 ml) at 25°C. under nitrogen was added a saturated solution of HBr in dioxane (2ml). The mixture was stirred for 10 minutes at which time the volatileswere removed completely in vacuo to give pure 3 (130 mg, 0.373millimoles, 86%) as a yellow solid: mp 99°-101° C.; R_(f) =0.78 in 9:1methylene chloride: ethyl acetate; IR (KBr) 3106, 3097, 3012, 2958,1724, 1592, 1510, 1339, 1068, 1053 cm⁻¹ ; ¹ H- NMR (acetone-d₆) δ 3.24(t, 2H, J=7.0 Hz), 3.46 (t, 2H, J=7.0 Hz), 4.28 (s, 2H), 7.98 (d, 1H,J=9.0 Hz), 8.48 (dd, 1H, J=2.5, 9.0 Hz); mass spectrum (FAB⁻) 268 (M -HBr)⁻, 233,199,183,109. Anal. Calcd. for C₁₀ H₉ BrN₂ O₅ S: C, 34.40; H,2.60; Br,22.89; N, 8.02; S, 9.18. Found C, 34.50; H, 2.60; Br, 22.70; N,7.88; S, 9.04.

4-(S-DNP)-mercaptobutanoic acid 10.

This compound was prepared as described above for 8, starting with4-mercaptobutanoic acid (1.80 g, 15 millimoles) giving 10 (3.21 g, 11.2millimoles, 75%) as a pale yellow solid: mp 126.5°-130° C.; IR (KBr)3528 (br), 3117, 3090, 2948, 1709, 1587, 1518, 1341 cm⁻¹ ; ¹ H- NMR(DMSO-d₆) δ 1.85 (m, 2H), 2.34 (t, 2H, J=7.0 Hz), 3.18 (t, 2H, J=7.5Hz), 3.78 (s, 1H), 7.95 (d, 1H, J=9.1 Hz), 8.41 (dd, 1H, J=2.4, 9.0 Hz),8.84 (d, 1H, J=2.4 Hz); mass spectrum (FAB⁻) 285 (M - H)⁻, 249,199,139.Anal. Calcd. for C₁₀ H₁₀ N₂ O₆ S: C, 41.96; H, 3.52; N, 9.79; S, 11.20.Found: C, 41.94; H, 3.50; N, 9.60; S, 10.97.

1-Diazo-2-oxo-5-(S-DNP)-mercaptopentane 11.

This compound was prepared as described above for 9, starting with 10(1.00 g, 3.49 millimoles), to give 11 (0.68 g, 2.20 millimoles, 63%) asa yellow solid: mp 84°-86° C.; R_(f) =0.52 in 9:1 methylene chloride:ethyl acetate; IR (KBr) 3106, 2939, 2866, 2104, 1626, 1589, 1513, 1343cm⁻¹ ; ¹ H- NMR (CDCl₃ δ 2.10 (m, 2H), 2.55 (t, 2H, J=5.7 Hz), 3.10 (t,2H, J=7.5 Hz), 5.29 (s, 1H), 7.74 (d, 1H, J=9.0 Hz), 8.38 (dd, 1H,J=2.5, 9.0 Hz), 9.06 (d, 1H, J=2.4 Hz); mass spectrum (FAB⁻) 310 (M⁻),249, 199, 139, 107. Anal. Calcd. for C₁₁ H₁₀ N₄ O₅ S: C, 42.58; H, 3.25;N, 18.06; S, 10.33. Found: C, 42.79; H, 3.39; N, 17.93; S, 10.14.

1-Bromo-2-oxo-5-(S-DNP)-mercaptopentane 4.

This compound was prepared as described above for 3, starting with 11(101 mg, 0.326 millimoles) to give 4 (116 mg, 0.32 millimoles, 98%) as ayellow solid: mp 87°-90° C.; R_(f) =0.78 in 9:1 methylene chloride:ethyl acetate; IR (KBr) 3113, 3009, 2953, 1723, 1592, 1512, 1343 cm⁻¹ ;¹ H- NMR (DMSO-d₆) δ 1.89 (m, 2H), 2.79 (t, 2H, J=7.1 Hz), 3.17 (t, 2H,J=7.4 Hz), 4.36 (s, 2H), 7.88 (d, 1H, J=9.1 Hz), 8.43 (dd, 1H, J=2.5,9.0 Hz), 8.85 (d, 1H, J=2.6 Hz); Anal. Calcd. for C₁₁ H₁₁ BrN₂ O₅ S: C,36.38; H, 3.05; Br, 2.00; N, 7.71. S, 8.83. Found: C, 36.57; H, 3.17;Br, 21,83; N, 7.49; S, 9.01.

5-(S-DNP)-mercaptopentanoic acid 12.

This compound was prepared as described above for 8, starting with4-mercaptopentanoic acid (2.00 g, 15 millimoles) giving 12 (4.20 g, 14.0millimoles, 93%) as a pale yellow solid: mp 159°-161° C.; IR (KBr) 3117,2948, (br), 1707, 1587, 1587, 1517, 1341 cm⁻¹ ; ¹ H- NMR (acetone-d₆) δ1.83 (m, 4H), 2.39 (t, 2H, J=6.8 Hz), 2.83 (s, 1H) 3.26 (t, 2H, J=7.0Hz), 7.95 (d, 1H, J=9.0 Hz), 8.45 (dd, 1H, J=2.5, 9.0 Hz), 8.96 (d, 1H,J=2.5 Hz); mass spectrum (FAB⁻) 299 (M-H)⁻, 199,183,141,113,109. Anal.Calcd. for C₁₁ H₁₂ N₂ O₆ S: C, 44.00; H, 4.03; N, 9.33. S, 10.68. Found:C, 43.83; H, 3.92; N, 9.42; S, 10.59.

1-Diazo-2-oxo-6-(S-DNP)-mercaptohexane 13.

This compound was prepared as described above for 9, starting with 12(1.00 g, 3.33 millimoles), to give 13 (0.70 g, 2.15 millimoles, 65%) asa yellow solid: mp 120°-122° C.; R_(f) =0.50 in 9:1 methylene chloride:ethyl acetate; IR (KBr) 3093, 2937, 2110, 1637, 1584, 1508, 1336, 1109cm⁻ ; ¹ H- NMR (CDCl₃) δ 1.83 (m, 4H), 2.39 (t, 2H, J=5.8 Hz), 3.04 (t,2H, J=7.4 Hz), 5.25 (s, 1H), 7.53 (d, 1H, J=9.0 Hz), 8.37 (dd, 1H,J=2.5, 9.0 Hz), 9.07 (d, 1H, J=2.5 Hz); mass spectrum (FAB⁻) 324, 307,267, 199, 183, 141, 109. Anal. Calcd. for C₁₂ H₁₂ N₄ O₅ S: C, 44.44; H,3.73; N, 17.28. S, 9.89. Found: C, 44.60; H, 3.74; N, 16.99; S, 9.65.

1-Bromo-2-oxo-6-(S-DNP)-mercaptohexane 5.

This compound was prepared as described above for 3, starting with 13(119 mg, 0.366 millimoles) to give 5 (0.127 g, 0.336 millimoles, 92%) asa yellow solid: mp 80°-83° C.; R_(f) =0.78 in 9:1 methylene chloride:ethyl acetate; IR (KBr) 3121, 2937, 2872, 1719, 1586, 1513, 1339, 1095,1053; ¹ H-NMR (acetone-d₆) δ 1.81 (m, 4H), 2.79 (t, 2H, J=6.6 Hz), 3,24(t, 2H, J=6.8 Hz), 4.18 (s, 2H), 7.93 (d, 1H, J=9.0 Hz), 8.45 (dd, 1H,J=2.5, 9.0 Hz), 8.95 (d, 1H, J=2.5 Hz); mass spectrum (FAB⁻) 378(M⁻),307, 263, 199, 183, 141, 109. Anal. Calcd. for C₁₂ H₁₃ BrN₂ O₅ S:C,38.21; H,3.47; Br,21.18; N,7.43; S,8.50. Found: C,38.40; H, 3.45;Br,21.34; N,7.34; S,8.46.

Antibody Modification

Affinity Labeling.

The strategy for introducing a thiol into the binding site of MOPC315 isoutlined in FIG. 3. These steps were carried out with the Fab fragment,generated by treating the reduced and alkylated IgA with papain followedby affinity chromatography on DNP-coupled Sepharose 4B (Goetzl (1970)Biochemistry 9:1267). The Fab fragment (at 10 μM) was treated witheither 1.5 equivalents of aldehydes 1 or 2 for one hour followed by 7.5equivalents of NaCNBH₃, in 0.2M sodium phosphate; pH 7.0, at 37° C. for20 hours, or with 1.3 equivalents of α-bromoketones 3, 4, or 5 in 0.1Msodium bicarbonate, pH 9.0; at 37° C. for 16 hours. In both cases, thelabeled Fab was purified by chromatography on Sephadex G-50 in 0.1Msodium phosphate, pH 7.3, and, after lyophilization, subsequent affinitychromatography on DNP-coupled Sepharose 4B (Goetzl, supra.) to removeunlabeled or non-specifically labeled Fab. The extent of derivatizationwas quantitated spectrophotometrically (for labels 1 and 2, λ_(max)=360nm, ε=12,800M⁻¹ cm⁻¹) and additionally, for labels 1 and 2, byincorporation of tritium from NaCNB³ H₃ (using 12 mCi/millimole NaCNB³H₃). With labels 2 and 4, over 90% of the label was incorporated (85%yield after affinity chromatography) (Table 1). In both cases, less than10% non-specific labeling occurred in the presence of 10 mM of thecompetitive inhibitor DNP-glycine.

                  TABLE 1                                                         ______________________________________                                        Incorporation of Affinity Labels into Fab                                              % Fab labeled   % Fab labeled                                        compd    (without DNP-glycine)                                                                         (with DNP-glycine)                                   ______________________________________                                        1        22              5                                                    2        95              10                                                   3         0              0                                                    4        85              0                                                    5        15              0                                                    ______________________________________                                    

Cleavage of Labels and Isolation of Stable S-Thiopyridyl Adducts.

Cleavage of the Fab affinity-labeled with 2 or 4 (2.0 mg) with 50 mMdithiothreitol in 2 mM EDTA, 0.1M sodium phosphate, pH 8.0 (2 ml) for 12hours at 37° C. afforded the free thiol The thiol-containing Fab wascollected by fast-desalting chromatography using a Pharmacia FPLC columnin 0.1M sodium phosphate, pH 7.3, directly into 1.0 ml of a 4.5 mMsolution of 2,2'-dithiodipyridine (0.1M sodium phosphate, pH 5.5,containing 15% acetonitrile). This mixture (final volume of 10 ml) wasallowed to react at 20° C. for 12 hours after which excess2,2'-dithiodipyridine was removed by exhaustive dialysis. The thiolatedantibody was derivatized in greater than 90% yield (65% recovery in thecase of 2) based on the absorbance of thiopyridine at 343 nm (ε=7060 M⁻¹cm⁻¹) (Grassetti et al. (1967) Arch. Biochem. Biophys. 119:41) aftercleavage with 10 mM dithiothreitol, and the protein absorbance at 280 nm(E₀.1% =1.37, MW=50,000 for Fab).

Determination of Modified Site.

Fab fragments labeled with 2 or 4 were subjected to tryptic digestionand peptide mapping in order to determine the selectivity of thiolincorporation. Fab was affinity-labeled in the presence of label 2 andNaCNB³ H₃ (12 mCi/millimole) or with ³ H-labeled 4 (4 mCi/millimole) asdescribed above. ³ H-labeled 4 was prepared as described for 4, startingwith 3,5-³ H-2,4-dinitrofluorobenzene. The labeled Fab (1.0 mg/ml) wasthen denatured in 8M urea, 0.1M Tris-HCl, pH 8.0, reduced with 20 mMdithiothreitol (1 hour at 37° C.) and alkylated with 60 mM iodoacetamide(1 hour at 37° C.). After dialysis against 7M urea, 20 mM Tris-HCl, pH8.0, the heavy and light chains were separated by anion exchangechromatography on a Pharmacia Mono Q FPLC column. Separation wasachieved at 1.0 ml/min in 7M urea, 20 mM Tris-HCl, pH 8.0, with a lineargradient of 40 mM to 200 mM sodium chloride over 30 min.

In the case of Fab labeled with 2, the heavy chain was found to containover 95% of the incorporated tritium; with Fab labeled with 4, over 95%of the tritium label was on the light chain. In each case, theradiolabeled chain was dialyzed against 2M urea/100 mM ammoniumbicarbonate, pH 8.2, and then treated with trypsin (1:25 w/wtrypsin:protein) in the presence of 0.1 mM CaCl₂ at 37° C. in the dark.After 8 hours, the reactions were quenched with 10% v/v acetic acid. Theradio-labeled peptides were purified by reverse phase HPLC with a 70min. linear gradient from 0 to 50% acetonitrile in water at 1.0 ml/min;(0.1% and 0.06% (v/v) trifluoroacetic acid was added to the water andacetonitrile, respectively.) Peptides were detected by their absorbanceat 214 nm, fractions were collected at 1.0 ml intervals, and aliquotswere counted for radioactivity (FIG. 4). Fractions containingradioactivity were re-chromatographed using a 70 min. linear gradientfrom 0 to 50% 2-propanol in water at 0.75 ml/min. The water and the2-propanol contained 0.1% and 0.06% trifluoroacetic acid, respectively.

The amino acid sequences of the pure peptides were determined on anApplied Biosystems 477A Protein Sequencer. With heavy chain obtainedfrom Fab labeled with 2, all of the detectable derivatization was onlysine 52H. With the light chain from Fab labeled with 4, all of thedetectable derivatization was on tyrosine 34L. These residues areidentical to those labeled by Givol and co-workers (Haimovich et al.(1972) Biochemistry 11:2389) with the reagents bromoacetyl-N^(e)-DNP-L-lysine and N-bromoacetyl-N'-DNP-ethylenediamine.

Ester Cleavage Using Thiol-Derivatized Antibody.

In addition to modifying the thiolated antibody selectively withcatalytic and other groups, the thiol itself can act as a nucleophile inthe thiolysis of appropriate substrate. DNP-containing esters 14 and 15(FIG. 5) were chosen as substrates for the reaction with thiolated Fablabeled with 2 or 4, respectively. The position of the cleavable linkagein affinity label 2 approximates that of the ester in the correspondingsubstrate 14, ensuring that the thiol is positioned appropriately in thecombining site to attack the ester. Likewise, the thiol in Fab labeledwith 4 should be positioned to attack ester 15. Moreover, thesesubstrates contain fluorescent coumarin leaving groups, which canreadily be detected at nanomolar concentrations.

Synthesis of Substrates.

(N-DNP)-3-Aminopropanoic acid 7-hydroxycoumarin ester 14.

A mixture of N-DNP-3-aminopropanoic acid (1.28g, 5 millimoles) and7-hydroxycoumarin (0.81 g, 5 millimoles) in phosphorus oxychloride (8ml) was heated at reflux under nitrogen for 2 hours. The mixture wascooled to room temperature and added to 60 ml of cold water. The brownsolid was filtered, washed with cold water and dried in vacuo.Trituration with acetone gave 14 (0.79 g, 2.0 millimoles, 40%) as alight brown solid; mp 155°-156° C., IR (KBr pellet) 3374, 3107, 2368,1750, 1722, 1624, 1532, 1426, 1349, 1159, 1127 cm⁻¹ ; ¹ H-NMR (DMSO-d₆)δ 3.04 (t, 2H, J=6.7 Hz), 3.90 (t, 2H, J=6.5 Hz), 6.48 (d, 1H, J=9.6Hz), 7.17 (d, 1H, J=8.4 Hz), 7.30 (s, 1H), 7.37 (d, 1H, J=9.7 Hz), 7.78(d, 1H, J=8.5 Hz), 8.07 (d, 1H, J=9.3 Hz), 8.29 (d, 1H, J=9.6 Hz), 8.87(s, 1H), 8.99 (m, 1H); mass spectrum (FAB⁺) 400(MH⁺). Anal. Calcd. forC₁₈ H₁₃ N₃ O₈ : C, 54,14; H, 3.26; N, 10.53. Found: C, 54.05; H, 3.26;M, 10.23.

2,4-Dinitrobenzoic acid, 7-hydroxycoumarin ester 15.

A mixture of 2,4-dinitrobenzoic acid (1.06 g, 5.0 millimoles) and7-hydroxycoumarin (0.81 g, 5.0 millimoles) in phosphorus oxychloride (4ml) was heated at reflux with a calcium sulfate drying tube for 40 min.The mixture was cooled to room temperature and added to water (40 ml).The brown solid was filtered, washed with cold water and dried in vacuoto give 15 (1.32 g, 3.7 millimoles, 74%) as a red-brown solid: mp200°-205° C.; IR (KBr) 3107, 3057, 2364, 1736, 1619, 1544, 1348, 1246,1122 cm⁻¹ ; ¹ H-NMR (DMSO-d₆) δ 6.53 (d, 1H, J=9.6 Hz), 7.34 (dd, 1H,J=2.2, 8.5 Hz), 7.47 (d, 1H, J=2.1 Hz), 7.89 (d, 1H, J=8.5 Hz), 8.12 (d,1H, J=9.6 Hz), 8.44 (d, 1H, J= 8.4 Hz), 8.78 (dd, 1H, J=2.2, 8.4 Hz),8.93 (d, 1H, J=2.2 Hz); mass spectrum (EI) 356 (M⁺), 195, 162, 134, 122.Anal. Calcd. for C₁₆ H₈ N₂ O₈ : C, 53.94; H, 2.26; N, 7.86. Found: C,52.18; H, 2.29; N, 7.51.

Ester Cleavage Assays.

Cleavage of DNP coumarin esters 14 and 15 by the thiol-containingantibodies was assayed in the presence of 0.1M NaCl, 50 mM sodiumphosphate, pH 7.0, with 24 μM dithiothreitol at 10° C. The release offree coumarin was quantitated fluorometrically, exciting at 355 nm andmeasuring emission at 455 nm. The S-thiopyridyl Fab was first dialyzedagainst assay buffer, 30 min. prior to the assays, an aliquot of thethiopyridyl Fab (0.15 mg in 0.19 ml) was reduced with 3.8 mMdithiothreitol at 20° C. For each assay, reduced, thiolated Fab (15 μgin 18.8 μl) was diluted with assay buffer (2.95 ml) so that the netconcentrations of thiolated Fab and of dithiothreitol were 0.1 μM and 24μM, respectively. After equilibrating, the substrate was added (30 μl ofa stock solution in acetonitrile) and the solution mixed for 10 sec.before monitoring fluorescence change. Antibody rates were corrected bysubtracting the rate of cleavage in the absence of Fab.

The antibody affinity-labeled with 2 was found to accelerate thecleavage of ester 14 by a factor of 6×10⁴ over the cleavage reaction inequimolar dithiothreitol. The reaction kinetics are consistent with theformation of a Michaelis complex (FIG. 6). The kinetic constants k_(cat)and K_(m) for the reaction are 0.87 min⁻¹ and 1.2 μM, respectively. Thethiolysis reaction was competitively inhibited ##STR1## by DNP-glycinewith a K_(i) of 8 μM. Neither the uncleaved affinity-labeled antibodynor the iodoacetamide-alkylated antibody accelerated the rate of thethiolysis reaction above the background rate. The stoichiometry ofproduct release corresponded to 1.0 coumarin to 1.0 Fab-SH. Addition ofhydroxylamine to the reaction buffer did not lead to multiple turnovers.However, the introduction of both a thiol and a general base into thelabel may lead to a catalytic system (Bruice (1959) J. Am. Chem. Soc.81:5444 and Street et al. (1985) J. Am. Chem.Soc. 107:7669).Interestingly, thiolated Fab labeled with bromoketone 4 did nothydrolyze DNP-acetate. Fluoroescence quenching experiments revealed thatthe derivatized Fab did not bind the substrate with appreciableaffinity, presumably as a result of steric congestion of the antibodycombining site.

Example 2

Introduction of Active Functionality by Linkage to Reactive Side ChainPreviously Introduced to Antibody Using Affinity Label.

The following experiment describes the post-translational introductionof a catalytic group into an antibody in close proximity to the antibodycombining site. The catalytic group was covalently attached to a thiolin the antibody MOPC 315 which was introduced as described in Example 1by the use of cleavable affinity label. The thiolated antibody describedin Example 1 was derivatized with an imidazole group providing aspecific catalyst for ester hydrolysis.

The antibody MOPC 315 binds substituted 2,4-dinitrophenyl (DNP) ligandswith association constants ranging from 5×10⁴ to 1×10⁶ M⁻¹. In order tosite-specifically incorporate a derivatizable thiol into the combiningsite of MOPC 315, a series of cleavable affinity labels was synthesized.These labels consist of the DNP group linked to electrophilic aldehydeor bromoketone groups through cleavable disulfide or thiophenyllinkages. Covalent attachment of the label to the antibody, followed bycleavage of the crosslink and removal of free ligand, results insite-specific incorporation of a free thiol into the antibody combiningsite (FIG. 3). It was determined that antibody affinity labeled with analdehyde labeling reagent in the presence of NaCNBH₃ provides, aftercleavage of the disulfide linkage, homogeneous antibody containing afree thiol at its binding site.

In order to derivatize this thiolated antibody with imidazole, thethiopyridyl disulfide adduct of the antibody (2.5 mg, 0.05 μmol) wastreated with 4-mercaptomethylimidazole (Street et al. (1985) J. Am Chem.Soc. 107:7669) (0.5 μmol) in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4(PBS) at 20° C. (FIG. 8). Incorporation of imidazole (FIG. 8) wasassayed by monitoring thiopyridone release spectrophotometrically at 343nm (ε=7060 M⁻¹ cm⁻¹). After 30 minutes, the reaction was complete, withquantitative release of thiopyridone. The mixture was dialyzedexhaustively against PBS and once against assay buffers. To verifyimidazole incorporation, a sample of the imidazole-antibody adduct (1.0mg) was reduced with 20 mM dithiothreitol and subjected to C18analytical reverse phase high performance liquid chromatography. (Alinear gradient was used from 0 to 20% acetonitrile in aqueous 0.1Mthiethylammonium acetate, pH 7.5 over 20 min., monitoring absorbance at230 nm. Under these conditions, a retention time of 4.6 min. wasobserved for 4-mercaptomethylimidazole). A peak in the elution profilewas observed with identical retention time to that for an authenticsample of 4-mercaptomethylimidazole.

The hydrolysis of coumarin esters 1a-1d (FIG. 9) by the semisyntheticantibody was assayed in the presence and absence of 1 μM derivatized Fabat varying pH, at 30° C. (Morpholineethanesulfonic acid (100 mM) as usedas the buffer in the range of pH 5 to 7. Sodium phosphate (100 mM) wasused in the range of pH 6 to 8 and tris-HCl (100 mM) was used in therange of pH 7 to 9.) The release of free coumarin was quantitatedfluorometrically, exciting at 355 nm and measuring emission at 455 nm.Antibody rates were corrected by subtracting the rate of cleavage in theabsence of antibody. From an Eadie-Hofstee plot of initial rate data,the kinetic constants, K_(m) and k_(cat), for the hydrolysis of ester 1bwere determined to be 1.9% 0.2 μM and 0.051% 0.005 min⁻¹, respectively(pH 7.0). The hydrolysis reaction is competitively inhibited byN-DNP-glycine with a K_(i) of 4% 1 mM (pH 7.0). This value is almostidentical to the K_(D) (5 μM) for the binding of N-DNP-glycine tounderivatized MOPC 315, (determined by fluorescence quenching in PBS).The catalytic activity of the imidazole-derivatized antibody shows a pHdependence consistent with a titratable residue with pKa 7.7% 0.2, whichis similar to the pKa of 4-methylimidazole, 7.5. At pH>8, the unmodifiedFab slightly accelerates the cleavage of ester 1b in a stoichiometricreaction, presumably involving modification of an amino acid side chainnear the binding site. However, this reaction is not observed in thecatalysis by the imidazole-derivatized antibody, where the reactiveantibody side-chain is probably inaccessible. The catalytic activity ofthe semisynthetic antibody is destroyed by treatment withdiethylpyrocarbonate, an imidazole-specific reagent. These data areconsistent with the presence of a catalytic imidazole acting either as ageneral base or directly as a nucleophile in the hydrolysis of ester 1b.The rate of the antibody-catalyzed reaction decreased when shorter orlonger esters were used as substrates (FIG. 9), presumably due to stericconstraints in the binding site with the shorter substrates and higherentropic barriers with the longer esters. The second-order rate constantfor the antibody-catalyzed reaction, k_(cat) /K_(m), was compared to therate constant for the reaction catalyzed by 4-methylimidazole at pH 7.The ratio [k_(cat) /K_(m) ]/k_(4-methylimidazole) gives an accelerationfactor of 1.3% 0.1×10³ for hydrolysis by the antibody.

Synthesis of Substrates

N-2,4-Dinitrophenyl (DNP) glycine, 7-hydroxycoumarin ester 1a.

A mixture of N-DNP-glycine (603 mg, 2.5 mmol) (Sigma) and thionylchloride (1.0 mL) in dry THF was stirred at room temperature undernitrogen for 12 h. The volatiles were removed in vacuo. Twice, dry THF(10 mL) was added and removed in vacuo to ensure complete removal ofexcess thionyl chloride. The residue was dissolved in dry THF (10 mL)and treated with 7-hydroxycoumarin (405 mgg, 2.5 mmol) followed bytriethylamine (0.70 mL, 5.0 mmol). The mixture was stirred at roomtemperature for 6 h when the solvent was removed in vacuo. The residuewas washed with cold water and dried in vacuo to give 1a (390 mg, 1.01mmol, 41%) as a yellow solid: mp 220°-222° C.; IR (KBr pellet) 3349,3100, 1729, 1624, 1525, 1398, 1349, 1237, 1117 cm⁻¹ ; ¹ H-NMR (DMSO-d₆)δ 4.75 (d, 2H, J=4.1 Hz), 6.50 (d, 1H, J=9.6 Hz), 7.23 (d, 1H, J=8.4Hz), 7.30 (s, 1H), 7.37 (d, 1H, J=9.7 Hz), 7.78 (d, 1H, J=8.5 Hz), 8.07(d, 1H, J=9.3 Hz), 8.29 (d, 1H, J=9.6 Hz), 8.87 (s, 1H), 9.10 (m, 1H);mass spectrum (FAB⁺) 386 (MH⁺). Anal. Calcd. for C₁₇ H₁₁ N₃ O₈ : C,52.99; H, 2.86; N, 3.64. Found C, 52.81: H, 2.92: N, 3.50.

N-DNP-3-aminopropanoic acid,7-hydroxycoumarin ester 1b.

A mixture of N-DNP-3-aminopropanoic acid (1.28 g, 5 mmol) and7-hydroxycoumarin (0.81 g, 5 mmol) in phosphorous oxychloride (8 mL) washeated at reflux under nitrogen for 2 h. The mixture was cooled to roomtemperature and added to 60 mL of cold water. The brown solid wasfiltered, washed with cold water and dried in vacuo. Trituration withacetone gave 1b (0.79 g, 2.0 mmol, 40%) as a light brown solid: mp155°-156° C., IR (KBr pellet) 3374, 3107, 2368, 1750, 1722, 1624, 1532,1426, 1349, 1159, 1127 cm⁻¹ ; ¹ H-NMR (DMSO-d₆) d 3.04 (t, 2H, J=6.7Hz), 3.90 (t, 2H, J=6.5 Hz), 6.48 (d, 1H, J=9.6 Hz)m 7.17 (d, 1H, J=8.4Hz), 7.30 (s, 1H, 7.37 (d, 1H, J=9.7 Hz), 7.78 (d, 1H, J=8.5 Hz), 8.07(d, 1H, J=9.3 Hz), 8.29 (d, 1H, J=9.6 Hz), 8.87 (s, 1H), 8.99 (m, 1H);mass spectrum (FAB⁺) 400 (MH⁺). Anal. Calcd. for C₁₈ H₁₃ N₃ O₈ : c,54.14; H, 3.26; N, 10.53. Found: C, 54.05; H, 3.26; N. 10.23.

N-DNP-aminovaleric acid, 7-hydroxycoumarin ester 1c.

A mixture of N-DNP-aminovaleric acid (566 mg, 2.0 mmol) (Sigma) and7-hydroxycoumarin (324 mg, 2.0 mmol) in methylene chloride (20 mL) wascooled to 0° C. Dicyclohexylcarbodiimide (454 mg, 2.2 mmol) was added.The mixture was stirred under nitrogen at 0° C. for 2 h and at roomtemperature for 24 h. The dicyclohexyl urea was filtered off and thefiltrate concentrated in vacuo. Trituration from 1:1 hexanes:ethylacetate afforded 1c (384 mg, 0.90 mmol, 45%) as a yellow solid: mp124°-125° C.: IR (KBr pellet) 3340, 3107, 2831, 1729, 1624, 1525, 1504,1419, 1349, 1258, 1159, 1124 cm⁻¹ ; H-NMR (CDCl₃) δ 1.60 (m, 2H), 1.95(m, 2H), 2.73 (t, 2H, J=6.7 Hz)m 3.50 (t, 2H, J=6.5 Hz), 6.41 (d, 1H,J=9.5 Hz), 6.94 (d, 1H, J=9.5 Hz), 7.04 (d, 1H, J=8.4 Hz), 7.26 (s, 1H),7.50 (d, 1H, J=8.4 Hz), 7.70 (d, 1H, J=9.5 Hz), 8.29 (d, 1H, J=9.5 Hz),8.59 (m, 1H)m 9.15 (s, 1H); mass spectrum (FAB⁺) 428 (MH⁺). Anal. Calcd.for C₂₀ H₁₇ N₃ O₈ : C, 56.21; H, 3.98; N, 9.84. Found: C, 55.88; H,3.88; N, 9.50.

N-DNP-aminocaproic acid, 7-hydroxycoumarin ester 1d.

This compound was prepared as described above for 1c, starting withN-DNP-aminocaproic acid (Sigma), to give 1d (36%) as a yellow solid: mp110°-112° C.; IR (KBr pellet) 3339, 3107, 2931, 1735, 1624, 1525, 1426,1335, 1278, 1117 cm⁻¹ ; ¹ H-NMR (CDCl₃) δ 1.60 (m, 2H), 1.85 (m, 4H),2.67 (t, 2H, J=7.2 Hz), 3.48 (t, 2H, J=6.4 Hz), 6.41 (d, 1H, J=9.5 Hz),6.94 (d, 1H, J=9.5 Hz), 7.04 (d, 1H, J=8.4 Hz), 7.26 (s, 1H), 7.50 (d,1H, J=8.4 Hz), 7.70 (d, 1H, J=9.5 Hz), 8.29 (d, 1H, J=9.5 Hz), 8.59 (m,1H), 9.15 (s, 1H); mass spectrum (FAB⁺) 442 (MH⁺). Anal. Calcd. for C₂₁H₁₉ N₃ O₈ : C, 57.14; H, 4.31; N, 9.52. Found: C, 56.99; H, 4.40; N,9.36.

Example 3

Hapten-dependant Preparation of Antibody Having Active Functionality inBinding Site

The following example demonstrates the preparation of an antibody havinga catalytic side chain in the binding site elicited by an antigen havingstructural features complementary to the desired side chain. Inparticular, antibodies are generated which catalyze the photocleavage ofthymine cyclobutane dimers, the predominant DNA photolesion produced byUV light (Patrick (1977) Photochem. Photobiol. 25:357-372 and Patrick(1977) Photochem. Photobiol. 25:373-384).

Organisms have evolved a number of mechanisms for the repair ofpyrimidine dimers, including the photoreactive enzyme DNA photolyasewhich cleaves thymidine dimers upon irradiation with visible light(Sutherland (1981) The Enzymes 14:481-515). Although the mechanism ofenzymatic repair remains unclear, photosensitizers such as indoles,quinones, and flavins appear to reversibly transfer an electron to oraccept an electron from the dimer, resulting in facile cleavage of theintermediate thymine dimer radicle (Rokita et al. (1984) J. Am. Chem.Soc. 106:4589-4595 and Jorns (1987) J. Am. Chem. Soc. 109:3133-3136).

The intent of the following experiment was to provide a photoreactingagent by preparing an antibody combining site specific for a thyminedimer and containing an appropriately positioned photosensitizer.Antibodies were generated against the polarized π system of a pyrimidinedimer and were found to contain a complementary tryptophan residue inthe combining site.

Antibodies were generated against a cis,synthymine dimer 2 (FIG. 10).The stereochemistry of the photocyclization reaction leading to 2 wascontrolled via dimerization of the N-1-carboxymethyl derivative, as theethylene glycol diester (Leonard et al. (1969) J. Am. Chem. Soc.91:5855-5862). Hapten 2 was synthesized by alkylation of thymine withchloroacetic acid in aqueous potassium hydroxide (Jones et al. (1973)Tetrahedron 29:2293-2296). Carboxymethylthymine was treated with oneequivalent of N,N'-carbonyldiimidazole (CDI) in dimethylformamide (DMF)followed by addition of one-half equivalent of ethylene glycol. Removalof solvent and trituration with water yielded the ethylene glycoldiester. The diester was dissolved in minimal DMF and photolyzed indegassed 10% aqueous acetone (1g/750 mL) through a Pyrex filter for 2hours using a 450 watt Hanovia medium pressure Hg immersion lamp.Removal of solvent and trituration with water afforded the thymine dimerethylene glycol diester as a single isomer (cis,syn). Basic hydrolysisand subsequent acidification to pH 2 gave crystallinecarboxymethylthymine-(cis,syn)-cyclobutane dimer 1. Activation with CDIin DMF followed by addition of glycine ethyl ester yielded thebis[ethylglycinate] adduct after removal of solvent and solvent gavehapten 2. N,N'-dimethylcarboxy-methylthymine(cis,syn)-cyclobutane dimerwas prepared by alkaline dimethylsulfate treatment (Wulff et al. (1961)Biochem. Biophys. Acta 51:332-339) of the carboxymethylthymine ethyleneglycol diester photoproduct followed by adjustment of the pH to 11 for 1hour and subsequent acidification to pH 2 to induce crystallization.

Dimer 2 was coupled via its N-hydroxysuccinimide ester to the carrierproteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH)and exhaustively dialyzed against 150 mM NaCl, 10 mM phosphate buffer,pH 7.5. Epitope densities ranged between 5 and 15. Balb/c mice wereimmunized with the KLH conjugate of 2 and antibodies were generated bystandard protocols (Jacobs et al. (1987) J. Am. Chem. Soc.109:2174-2176). IgG was purified from ascites fluid by affinitychromatography on protein A-coupled Sepharose 4B and judged homogeneousby sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli(1970) Nature 227:680-685).

Solutions of carboxymethylthymine dimer 1 were irradiated with 300 nmlight in the presence of antibody; and dimer cleavage was assayedspectrophotometrically as follows. Deoxygenated solutions of thyminedimer (5-300 μM) and antibody (3 μM) in 100 mM NaCl, 10 mM phosphatebuffer, pH 7.5 were irradiated at 18°-20° C. in a 1 mL quartz cuvette.All photolysis experiments were performed with a 1000 W high pressureHg-Xe lamp equipped with a Photon Technologies International, Inc. ModelQ5001 Quantacount electronic actinometer and a diffraction gratingmonochromator blazed at 500 nm. A bandpass of 3 nm was used for bothkinetic and photon counting experiments. Kinetic experiments wereperformed at 300 nm with an incident flux of 1.26×10⁻⁷ einsteins min⁻¹.Monomer formation was assayed spectrophotometrically at fixed timeintervals (λ_(max) =272 nm, log ε=3.90). Protein molarity was determinedby absorbance at 280 nm by using ε⁰.1% =1.37 and a molecular weight of150 kD for IgG.

Five out of six antibodies (IgG) were found to sensitize thephotocleavage. One of these, antibody 15F1-3B1 was studied further. Highpressure liquid chromatography confirmed that the reaction product wasthymine monomer. The absorption and fluorescence spectra of the antibodyremained unchanged upon photolysis, indicating that photodegradation ofthe protein was negligible. The kinetics of the antibody-catalyzedreaction are consistent with the Michaelis-Menten rate expression (FIG.11): ##STR2## The kinetic constants k_(cat) and K_(m) are 1.2 min⁻¹ and6.5 μM, respectively (k_(cat) /K_(m) =1.8×10⁵ M⁻¹ min⁻¹). The k_(cat)value observed is comparable to the turnover number for Escherichia coliDNA photolyase of 3.4 min⁻¹ (Jorns et al. (1985) Biochemistry24:1856-1851). The reaction is first order in light between incidentintensities of 5×10⁻⁸ and 1.6×10⁻⁷ einsteins min⁻¹. Consequently, thek_(cat) obtained is not optimal, and irradiation at higher flux shouldincrease k_(cat) until light saturation occurs (Eker et al. (1986)Photochem. Photobiol. 44:197-205). The first-order rate constant forunsensitized dimer cleavage is 5.5×10⁻³ min . Hapten 2 is also readilycleaved by the antibody; however the K_(m) for this substrate is too lowto be conveniently measured by direct spectrophotometric assay (<1 μM).The photocleavage of the corresponding N,N'-dimethyl substrate is notsensitized by the antibody at substrate concentrations of up to 1.8 mM,which is consistent with the high specificity of antibody-ligandbinding. In addition, the dinitrophenyl specific IgA MOPC 315, whichcontains a binding-site tryptophan, and the phosphorylcholine specificIgA MOPC 167 do not catalyze the photocleavage of thymine dimer underidentical reaction conditions (FIGS. 11(a) and 11(b).

The wavelength dependence of the quantum yield of theantibody-sensitized photolysis of hapten 2 reveals a shoulder at 300 nm(FIG. 12(a)). Antibody fluorescence is also quenched in the presence ofthymine dimer (FIG. 12(b)). It should be noted that the percentage offluorescence quenched upon ligand binding does not reflect the totalnumber of tryptophan residues in the protein due to environmentaleffects on Φ_(F). These observations indicate that a combining sitetryptophan is photosensitizing dimer cleavage.

The quantum yield of the antibody-catalyzed reaction (Φ_(R), 300) is0.08. The antibody was oxidized with N-bromosuccinimide in 8M urea, pH4.5, revealing 10 tryptophan residues per Ig (Spande et al. (1967)Methods in Enzymology 11:498-506). Assuming that each binding sitecontains only one tryptophan allows the calculation of an approximatequantum yield of 0.4 for photocleavage of bound dimer (based onformation of monomer). Pulse radiolysis experiments have shown thatdimethyl and tetramethylthymine dimer radical anions decay to monomerwith a frequency of 0.05 (Lamola (1974) Mol. Photochem. 4:107-133).Since the quantum yield of the antibody-sensitized reaction issignificantly higher than 0.05, the antibody appears to partition thebreakdown of the intermediate radical anion.

Example 4

Hapten-dependent Preparation of Antibody Having Active Functionality inBinding Site.

The following example demonstrates the preparation of an antibody havinga catalytic side chain in the binding site elicited by an antigen havingstructural features complementary to the desired side chain. Inparticular, antibodies are generated which catalyze the elimination ofhydrogen fluoride from a β-fluoroketone.

The intent of the following experiments was to provide an antibodycontaining a catalytic side chain (such as a glutamate or aspartate)capable of abstracting a proton from a β-fluoroketone substrate bound inthe antibody combining site. It has been demonstrated that haptens witheither a positive or negative charge will elicit a complementarynegatively charged or positively charged amino acid side chain,respectively, in an antibody combining site. Nisinoff et al. (1975) TheAntibody Molecule, Academic Press, pp 23-27. In order to introduce acarboxylate in the combining site of an antibody to the fluoroketonesubstrate (FIG. 13), antibodies were generated to the correspondinghapten which contains an ammonium ion. This ammonium ion is positionedso as to elicit a carboxylate in the antibody that will be in closeproximity to the abstractable proton in the fluoroketone substrate. Inaddition, the pKa of the carboxylate should increase when the substrateis bound (the carboxylate is no longer stabilized by a salt-bridge)thereby making it a better base for proton abstraction. Antibodies weregenerated against benzylamine.

Synthesis of Hapten and Substrate

N(p-nitrobenzyl)δ-aminovaleric acid.

1.55 g (10 mmol) of δ-aminovaleric acid potassium salt was dissolved in40 ml of methanol and adjusted to pH 6-7 with conc. methanolic HCl. Theprecipitated potassium chloride was filtered off and washed with 10 mlof methanol. To the filtrate was added 1.51 g (10 mmol) ofp-nitrobenzaldehyde and 437 mg (7 mmol) of sodium cyanoborohydride. Themixture was stirred for 16 h at rt. The reaction was quenched by adding5 ml of conc. HCl. The mixture was evaporated, redissolved in 100 ml ofwater and extracted with 2×50 ml of dichloromethane (extraction ofunreacted or reduced aldehyde). The aqueous phase was concentrated andloaded on a DEAE-sephadex column (3×30 cm, conditioned with 0.5Mtriethylammonium bicarbonate pH 8.2 and subsequently washed with water).The product was eluted with a gradient of A) water and B) 0.5Mtriethylammonium bicarbonate pH 8.2, B=0-50% (500 ml). The fractionswere monitored by tlc (silicagel/n-BuOH:AcOH:H₂ O)=4:1:1). The fractionscontaining pure product were combined and evaporated. The residue wasfreed from triethylammonium bicarbonate by ion exchange chromatographyon Dowex 50 (H⁺ -form). After washing the adsorbed product with 100 mlwater, it was eluted with 1M ammonia (100 ml). Evaporation andcrystallization from water/acetone afforded 799 mg (32%) of pureproduct. mp. 123°-125°; 1H-NMR (200 MHz,D₂ O):8.11 (d, J=8.8/2H), 7.52(d, J=8.8/2H), 4.19 (s/2H), 2.97 (m(t)/2H), 2.05 (t, J=7/2H), 1.57-1.45(m/4H); 13C-NMR (50 MHz, D₂ O+DMSO=37.9): 181.6, 175.7, 147.2, 137.2,129.9, 123.3, 49.1, 46.4, 35.8, 24.4, 21.7; FAB⁺ -MS: 253 (M+1)⁺ ; UV(H₂ O) 263 nm (=10100); EA (C₁₂ H₁₆ N₂ O₄) calc: C 57.13 H 6.39 N 11.10,found: C 56.93 H 6.46 N 10.99.

N-methyl N-(p-nitrobenzyl)δ-aminovaleric acid.

To a solution of 300 mg (1.19 mmol) of N-(p-nitrobenzyl)δ-aminovalericacid in 6 ml of water were added 450 μl of a 37% aqueous formaldehydesolution (6.1 mmol) and 123 mg of sodium cyanoborohydride (1.96 mmol).This mixture was stirred for 45 min at rt. After quenching with 6 ml of1M hydrochloric acid the solution was directly adsorbed on a Dowex 50(H⁺ -form) ion exchange column (2×10 cm). The column was subsequentlywashed with 150 ml water and the product eluted with 0.7M ammonia. Afterevaporation, the remaining oil was further purified by silicagelchromatography (1×15 cm, methanol:dichloromethane=1:1). The productcontaining fractions (Rf=0.22) were collected and the solvent removed invacuo. 270 mg (85% of product in form of a colorless oil was isolated.Attempts at crystallization failed. 1H-NMR (200 MHz,D₂ O):8.10(d,J=8.7/2H), 7.46 (d, J= 8.7/2H), 3.83 (s/2H), 2.59 (m(t)/2H), 2.28(s/3H), 2.03 (m(t)/2H), 1.49-1.39 (m/4H); 13C-NMR (50 MHz, d⁶ -DMSO):175.3, 147.8, 146.4, 129.4, 123.3, 60.6, 56.6, 41.8, 34.1, 26.3, 22.5;FAB⁺ -MS:267 (M+1)⁺. EA (C₁₃ H₁₈ N₂ O₄. 0.H₂ O) calc. C 55.26 H 7.06 N9.91, found: C 55.34 H 6.88 N 9.89.

BSA-conjugate of N-methyl N-(p-nitrobenzyl)δ-aminovaleric acid.

A solution of 27 mg (0.1 mmol) N-methyl N-(p-nitrobenzyl)δ-aminovalericacid in 1 ml water was adjusted to pH 2 by addition of 6 drops of 1Maqueous HCl. This solution was subsequently lyophylized and dried on HVfor 72 h at rt. The residue was dissolved in 1.5 ml DMF and 62 mg (0.54mmol) N-hydroxysuccinimide and 166 μl 1,3-dicyclohexylcarbodiimide wereadded. After 20 h stirring at rt, the mixture was centrifuged and thesupernatant added to a solution of 30 mg BSA in 4 ml 0.75 mM aqueoussodium carbonate pH 9.3. The pH was periodically controlled and adjustedto 9.3 by addition of 0.1M aqueous sodium hydroxide. After 20 h stirringat rt the solution was extensively dialyzed against 150 mM sodiumchloride, 10 mM sodium phosphate buffer pH 7.4 (3×2 lt). Allprecipitated material was centrifuged. The protein concentration of thefinal solution was determined to be 3.41 mg/ml. The grade ofderivatization with hapten was assayed by measuring selectively theremaining free lysine amino groups on a aliquot of the protein solution.Calculating with 59 lysines in BSA an epitope density of 5 was measured.

KLH-conjugate of N-methyl N-(p-nitrobenzyl)δ-aminovaleric acid.

This was achieved in an analogous way as for the BSA-conjugate, usingKLH as the carrier protein. The concentration of the final proteinsolution (Lowry) was determined to be 0.62 mg/ml with an epitope density(Habeeb assay, calculating for 34 lysines in KLH) of 18.

(R,S) 4-Hydroxy-4-p-nitrophenylbutan-2-one.

To a 250 mL round-bottom flask cooled in an ice bath, 120 mL of acetoneand 10 g (66.2 mmol) of p-NO₂ benzaldehyde were added. Dropwise additionof 12 mL of a 1% NaOH solution effected a change in color from yellow todeep orange. Stirring was continued at 0° C. for 15 minutes followed byaddition of 0.5N HCl to pH 7.00. Volatiles were removed by rotaryevaporation leaving a black oil containing water. The oil was dissolvedin water and extracted several times with diethyl ether. The etherextracts were combined and dried with MgSO₄. The ether was removed byrotary evaporation and the orange oil was purified by flashchromatography on 300 g of silica gel, eluting with 1:1 hexanes:ethylacetate. The fractions containing product (R_(f) =0.4) were combined andthe solvent was removed by rotary evaporation to afford 10.23 g (74%) ofproduct:mp 59°-61° C.; 1H-NMR (250 MHz, CDCl₃): δ 2.23 (s, 3H); 2.88 (d,2H, J=6.2); 3.90 (d, 1H, 3.4); 5.27 (m, 1H); 7.54 (d, 2H, J=8.7); 8.16(d, 2H, J=8.7); UV λ_(max) =275 (log ε=5.00) mass spectrum (EI) 209(mt),191 (M-H₂ O). Anal. calcd. for C₁₀ H₁₁ N₁ O₄ : C, 57.44, H, 5.26, N,6.69. Found: 57.23, 5.26, 6.79.

(R,S) 4-Fluoro-4-p-nitro phenylbutan-2-one.

To a 3-neck 100 mL round-bottom flask 1.42 g (6.79 mmol) of4-hydroxy-4-p-nitrophenyl-butan-2-one and 70 mL of freshly distilledmethylene chloride were added. At -78° C. 0.90 mL (6.79 mmol) ofdiethylaminosulfurtrifluoride was added dropwise over 10 minutes.Stirring was continued for an additional 10 min. after which thereaction was quenched with aqueous 0.1M citrate pH 5.00 buffer. Theorganic layer was quickly separated and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the methylene chloride was removed by rotaryevaporation affording 0.99 g (69%) of an orange oil. 'H NMR (250 MHz,CDCl₃): δ 2.23 (s, 3H); 2.85 (m, 1H); 3.22 (m, 1H); 6.15 (m, 1H); 7.52(d, 2H, J=8.9); 8.23 (d, 2H, J=8.3). UV λ_(max) = 282 (log ε=3.99). Massspectrum (EI) 211 (mt), 196 (m-methyl). Anal. calcd. for C₁₀ H₁₀ N₁ O₃F₁ : C, 56.87; H, 4.74; N, 6.61. Found: 56.74, 4.83, 6.54.

Hapten (N-methyl N-(p-nitrobenzyl)δ-aminovaleric acid) was coupled tothe carrier proteins bovine serum albumin (BSA) and keyhole limpethemocyanin (KLH) and exhaustively dialyzed against 150 mM NaCL, 10 mMphosphate buffer, pH 7.5. Epitope densities ranged between 5 and 15.Balb/c mice were immunized with the KLH conjugate of N-methylN-(p-nitrobenzyl)δ-aminovaleric acid and antibodies were generated bystandard protocols (Jacobs et al. (1987) J. Am. Chem. Soc.109:2174-2176). Ig was purified from ascites fluid by SDS polyacrylamidegel electrophoresis (Laemmli (1970) Nature 227:680-685).

Solutions of (RS) 4-fluoro-4-p-nitro phenylbutan-2-one were incubatedwith 2 μM Ig at 37° C. in 10 mM Bis-tris-HCl, 100 mM NaCl pH 6.0, atsubstrate concentration between 50 and 500 μM. Protein molarity wasdetermined by absorbance at 280 nm using ε⁰.1% =1.37 and a molecularweight of 150 kD for IgG. Elimination was followedspectrophotometrically. Four out of the six antibodies isolated (IgG)were found to catalyze the elimination above the background rate. One ofthese antibodies was characterized further. Eadie-Hofstee plots ofinitial rate data afforded a k_(cat) value of 0.188 min⁻¹ and Km valueof 182 μM. The kinetics of the reaction are consistent with the simpleMichaelis-Menten rate expression: ##STR3## These values afford a k_(cat)/k_(uncat) of 1480 at pH 6.0.

It was also demonstrated that the antibody-catalyzed reaction iscompetitively inhibited by hapten. Values of K_(i) were determined at 10mM Bis-tris-HCl, 100 mM NaCI, pH 6.0 (37° C.). Substrate concentrationswere 200 and 500 μM, Ig concentration was 2 μM, hapten concentration was200 mM to 1 μM. Inhibition was demonstrated to be competitive with aK_(i) =290 nM.

The pH dependence of k_(cat) was also determined under the same reactionconditions. Values of k_(cat) are as follows: k_(cat) =0.083 min⁻¹ at pH5.5, k_(cat) =0.11 at pH 5.75, k_(cat) =0.188 at pH 6.0, k_(cat) =0.3 atpH 6.5 and k_(cat) =0.43 at pH 7.5. A plot of k_(cat) vs. k_(cat) [H⁺ ]gives a pKa of 6.1 for the active site base. It is very likely that thiscorresponds to a high pKa carboxylate based on the strategy forgenerating the antibody. Chemical modification experiments withdiazoacetamide support this hypothesis.

Example 5

Modification of Antibody S107 by Site-Directed Mutagenesis.

Monoclonal antibody S107, which is specific for choline phosphodiesters,is modified by site-directed mutagenesis to introduce activefunctionalities, including nucleophiles, chelated metals, and cofactors,as follows.

Antibody S107 has a number of features which make it useful as acatalytic antibody. First, S107 has been demonstrated to catalyze arylcholine carbonate hydrolysis by transition state stabilization. Second,the x-ray crystal structure of a homologous antibody McPC603 has beensolved (Padlan et al. (1985) Ann. Instit. Pasteur Paris 136:271-290).Third, a wide variety of substances to test hydrolytic and redoxreactions are easily synthesized, and fourth, the entire heavy and lightchain genes have been cloned and are available on the eukaryoticexpression vector pSV2-S107 (Tucker et al. (1979) Science206:1303-1306).

Antibody S107 includes an arginine residue at position 52 in the heavychain (Arg 52_(H)) and a tyrosine residue at position 33 in the heavychain (Tyr33_(H)). The crystal structure of the antibody shows that bothresidues appear to participate in stabilizing the tetrahydralnegatively-charged transition state in ester hydrolysis.

Tyrosine 33_(H) is changed to histidine, aspartate, and glutamate to actas general bases. Arginine 52 is replaced by cysteine which couldpotentially participate directly in catalysis or be modified withanother active group via disulfide exchange.

In order to carry out mutagenesis, pSV2-S107 was initially digested withthe restriction enzyme BAM H1 in an attempt to directly isolate the 1.2kilobasepair (kb) tragment containing the VDJ1 gene (FIG. 14). Due tothe multiple Bam H1 sites present in the plasmid, however, the fragmentcould not be cleanly isolated To circumvent this problem, the plasmidPSV2-S107 was digested with Eco R1 and Pvu 1 and the resulting 5 kbfragment was isolated via agarose gel electrophoresis. This fragmentwhich contains the heavy chain variable region coding sequences wassubcloned into the plasmid pT7-3. Restriction mapping confirmed that thedesired insert was present in the pT7-S107 DNA. Plasmid pT7-S107 wasdigested with Bam H1 and the 1.2 kb fragment was isolated via agarosegel electrophoresis. This fragment containing the VDJ1 gene was nowreduced to a suitable size for cloning into bacteriophage M13. Clearplaques were grown on 2YT growth medium and single stranded M13-S107 DNAwas isolated by a standard bacteriophage preparation. The sequence ofthe VDJ1 gene was determined using the dideoxy chain terminationsequencing method developed by Sanger. Oligonucleotide directedmutagenesis was performed using the method developed by Eckstein.Mutants were sequenced to be sure that the desired mutations hadoccurred. Double stranded DNA was digested with Bam H1 and the mutant1.2 kb fragments were inserted into PT7-S107.

The final step in the cloning strategy is to isolate the mutant heavychain gene (VDJ1) from the pT7-S107 plasmid and insert it into theparent vector PSV2. The resulting clones will possess mutant antibodygenes which will be expressed in appropriate eukaryotic cells, and themutant antibodies isolated. These antibodies will have the ability tocatalyze the cleavage of p-nitrophenylcarbonate esters. Potentialcatalytic groups can be introduced via selective derivatization of thecysteine 52 mutant.

The Cys 52_(H) mutant are further derivatized with the catalytic andchemically-reactive functionalities shown in FIG. 15. These derivativesare capable of promoting redox reactions, hydrolytic reactions, andtransamination reactions.

A Cys 52 mutant derivatized with a thiol containing-pyridoxamineprovides a catalytic antibody for promoting chiral transaminationreactions on a keto acid choline substrate.

A Cys S107 mutant derivatized with redox active metals such as therhodium phosphine ligand promotes hydrogenation reactions on a cholinesubstrate. The rhodium phosphine complex is stable in aqueous bufferedsolutions.

Derivatizations are carried out by disulfide exchange reactions usingeither thiopyridyl activated antibodies or thiopyridyl activatedcatalytic or reactive groups. Alternatively, thiol-containing antibodieswill be derivatized by selective alkylation by a catalytic or reactivegroup containing an electrophile such as an aldehyde or a bromoketone.

Example 6

Preparation of Hybrid Fv Fragment Based on MOPC315

A hybrid Fv fragment of the dinitrophenyl-binding immunoglobulin A,MOPC315, was produced by reconstituting recombinant V_(L) produced in E.coli with V_(H) derived from the antibody. A His residue was substitutedfor a Tyr at position 34 of V_(L), in order to introduce a catalyticimidazole into the combining site for the hydrolysis of esters. The Hismutant Fv accelerates hydrolysis of the 7-hydroxycoumarin ester of5-(2,4-dinitrophenyl)-aminopentanoic acid 90,000-fold compared to thereaction with 4-methyl imidazole at pH 6.8. The initial rate of estercleavage catalyzed by the mutant Fv is forty-five times faster than thatof the wild type Fv. The hydrolysis of aminopropanoic and aminohexanoichomologs are not significantly accelerated. These results demonstratethat a single deliberate amino acid change can introduce significantcatalytic activity into an antibody combining site and that chemicalmodification data can be used to locate potential sites for theintroduction of catalytic residues.

The well characterized antibody MOPC315 is secreted by a mineral-oilinduced murine myeloma cell line of the same designation, and binds avariety of DNP ligands with association constants of 10³ -10⁷ M⁻¹.MOPC315 IgA can be proteolyzed with pepsin to yield functional Fab' orFv fragments The Fv fragment (26 kD) is a heterodimer consisting of twopeptides, V_(H) (14 kD) and V_(L) (12 kD), and contains all thesequences necessary for folding of the binding domain and recognition ofthe DNP hapten. Although the atomic coordinates for MOPC315 have notbeen determined, magnetic resonance spectroscopy (Dower et al., inBiological Applications of Magnetic Resonance (R. G. Schulman, ed.,Academic Press, New York, 1979) p.271; Dwek et al. (1977) Nature 266:31;Leatherbarrow et al. (1982) Biochemistry 21:5124; and Gavish et al.(1979) Molecular Immunology 16:957) and affinity labelling (Haimovich etal. (1972) Biochemistry 11:2389) studies have provided some informationconcerning binding site structure.

Imidazole catalyzes the hydrolysis of carboxylate esters in aqueoussolutions. The objective of this example is to substitute a His residue(having an imidazole side chain) at the appropriate position in thecombining site of MOPC315 to produce a catalytic antibody with specifichydrolytic activity towards DNP-containing esters. In the absence of acrystal structure for MOPC315, data from chemical modificationexperiments were used to target residues for substitution with His. Inspite of the fact that there are 14 potentially reactive side chains inthe hypervariable region (2 His, 2 Lys, 3 Arg and 7 Tyr), DNP-containingaffinity labels alkylate primarily two residues, Tyr 34_(L) and Lys52_(H) (Pollack et al. (1988) Science 242:1038. The reactivity of eachresidue strongly depends on the number of atoms between the DNP ring andthe electrophilic carbon of the affinity reagent; Tyr 34_(L) isalkylated most efficiently by 4, while Lys 52_(H) reacts with 5 (FIG.16). Cleavable cross-linking agents were used to introduce a thiol(Example 1), and subsequently a catalytically active imidazole (Example5) into the MOPC315 combining site at position 52_(H). Tyr 34_(L) waschosen for mutagenesis since it appeared that a His 34_(L) side chainwould be well situated to catalyze the hydrolysis of esters 1-3 (FIG.16) (pollack et al. (1988) supra, and Pollack et al. (1989 ) J. Am.Chem. Soc. 111:1929). The role of Tyr 34_(L) in DNP binding has not beenclearly established. Affinity labelling of the Tyr hydroxyl to form the2-keto-5-thiol-pentyl ether prevents binding of ligands (Pollack et al.(1988) supra), perhaps by blocking the entrance to the site, whilenitration has no effect (Leatherbarrow et al. (1982) supra, Gavish etal. (1979) supra) Tyr 34_(L) is conserved in murine λ light chains andis found frequently in κ chains, and in heavy chains at the analogousposition (position 33). Phenylalanine (Phe) was also substituted atposition 34_(L) to assess the contribution of the Tyr hydroxyl group tohapten binding.

In order to generate mutant MOPC315 combining sites, the V_(L) portionof the Fv domain of the antibody was expressed in E. coli. A hybrid Fvwas then produced in which recombinant V_(L) produced in E. coli wasreconstituted with V_(H) derived from MOPC315 IgA. The protein sequenceof the V_(L) peptide (residues 1-115 (Hochman et al. (1973) Biochemistry12:1130; Hochman et al. (1977) ibid 15:2706)) was derived from a cDNAsequence of MOPC315 light chain mRNA (Kabat et al. Sequences of Proteinsof Immunological Interest, 4th Edition (U.S. Dept. of Health and HumanServices, 1987); Bothwell et al. (1982) Nature 298:380). The proteinsequences was converted to a DNA sequence and restriction enzymerecognition sites were incorporated. Otherwise, the most frequentlyoccurring E. coli codons were used. A gene consisting of these sequenceswas constructed from four restriction fragments (EcoRI-BstEII,BstEII-AvaII, AvaII-BssHII, BssHII-HindIII, denoted by the arrows abovethe sequence in FIG. 17). Complementary synthetic oligonucleotides 78 to99 bases in length were phosphorylated, annealed, and ligated into aderivative of M13mp18, either singly or as a pair. The cloned segmentswere sequenced and assembled into the full length gene in several steps.Cassette and primer mutagenesis (Nakamaye et al. (1986) Nucl. Acid Res.14:9679; Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488) were used tomake subsequent modifications, which resulted in the deletion of theEcoRI site.

The synthetic V_(L) genes were expressed as a fusion with the cII geneof coliphage λ (Nagai, et al. (1984) Nature 309:810; Nagai et al. (1987)Meth. Enzymol. 153:461) as illustrated in FIG. 18. A synthetic duplexencoding the Factor Xa recognition and cleavage sequence, IleGluGlyArg,was inserted in-frame at the 5' end of the V_(L) gene in M13. Theβ-globin gene in the expression vector pLcIIFXβ(nic⁻) (Nagai et al.supra) was excised by digestion with BamHI and HindIII and replaced bythe hybrid FXVL sequence. The Tyr 34 mutations (Tyr34(TAT)→His(CAT),Phe(TTT) were made in the V_(L) gene in M13 by the method of Kunkel(Nakamaye et al. supra) and then transferred into pLcIIFXVL bysubstitution of the BstEII-HindIII fragment.

The N-terminal extension of the cII-V_(L) hybrid was removed bysite-specific cleavage with Factor Xa (Fujikawa et al. (1972)Biochemistry 11:4882). The liberated V_(L) peptide was reconstitutedwith antibody-derived V_(H), and the resulting Fv was purified by gelfiltration and affinity chromatography. Antibody-derived V_(H) wasprepared by denaturing ion exchange chromatography of authentic MOPC315Fv, obtained from pepsin digestion of purified IgA and subsequentaffinity chromatography. Pepsin treatment affords two major V_(H)peptides and a small amount of incompletely digested V_(H) -containingfragments with copurify. Reconstitution experiments carried out in theabsence of V_(L) peptide afforded no DNP-binding or ester hydrolysisactivity.

SDS-PAGE analysis of various stages in the production of mutant Fvproteins was performed. The results are presented in FIG. 19. Lanes 1and 2: induced E. coli MZ1(pLcIIFXVL(Y34F) and (Y34H)); lanes 3 and 4:solubilized inclusion bodies; lanes 5 and 6: purified fusion proteins;lanes 7 and 8: Factor Xa cleaved and partially purified V_(L) peptides;lanes 9 and 10: reconstituted and affinity purified Fv(Y34F_(L)) andFv(Y34H_(L)); lane 11: Fv from proteolysis of MOPC315 IgA. The bandsabove V_(L) and V_(H) are completely digested V-containing heavy chainfragments which copurify with V_(H).

Gel filtration analysis of Fv(Y34H_(L)) was performed. The results arepresented in FIG. 20. Inclusion bodies were obtained from induced E.coli MZ1 harboring pLcIIFXLV(Y34H or Y34F) (Nagai et al. supra),solubilized in 8M urea, reduced with 20 mM dithiothreitol overnight at4° C., and chromatographed on a 1×30 cm column of S-sepharose in 8Murea, 20 mM Na-MES, pH 6.0, using a 0 to 200 mM NaCl gradient (yield: 10to 30 mg of purified protein per liter of induced cells (ε₂₈₀(1mg/ml)=1.0 (Hochman et al. supra)). The purified fusion proteins wereoxidized in air 30 hours, 4° C. in 2.5M urea, 20 mM tris-Cl, pH 8.4, andthen exhaustively dialyzed against 20 mM tris-Cl, pH 8.4, at 4° C. Threemg of fusion protein in 15 ml of 20 mM tris-Cl, pH 8.4, 1 mM CaCl₂, 30μg/ml kanamycin sulfate, was incubated for 30 hours, 37° C. with 600micrograms (μg) of bovine Factor X (prepared from the BaSO₄ eluate ofbovine plasma (Nagai et al. (1984) supra; Nagai et al. (1987) supra;Fujikawa et al. (1972) supra)(Sigma)) activated in situ with 2.5 μgcrude Russell's viper venom (Sigma). Crude V_(L) was made 100 mM in NaCland passed through a 2 ml bed of Q-sepharose, dialyzed against distilledwater, lyophilized, and dissolved in 8M urea, 20 mM potassium phosphate,pH 6.0. Fv(315) was purified from ascites fluid from MOPC315 myelomagrown in Balb/c mice as previously described (Eisen et al. (1968)Biochemistry 7:4126; Hochman et al. (1973) supra; Hochman et al. (1977)supra) except the gel filtration step after pepsin cleavage was omitted.V_(H) peptide was separated from V_(L) by ion exchange chromatography(Pharmacia mono Q 10/10 column) in 20 mM tris-Cl, pH 8.0, 8M urea, usinga 0 to 250 mM NaCl gradient. Cleaved V_(L) (0.1 to 1 mg) and equimolarV_(H) were rapidly diluted ten-fold from 8M urea into 100 mM potassiumphosphate, pH 6.0 (final protein concentration=50 μg/ml) and allowed tostand 30 minutes 25° C. Active Fv was adsorbed onto DNP-lysine sepharose(Hochman et al. (1973) supra: Hochman et al. (1977) supra: Inbar et al.(1971) J. Biol. Chem. 246:6272), washed with 100 mM NaCl, 50 mM tris-Cl,pH 8.0 and eluted with a small volume of 30 mM DNP-glycine, pH 8.0. Theeluate was dialyzed against 100 mM potassium phosphate, pH 6.8. Overallyield from the fusion protein is 5-20%. The yield of reconstitution is20-30% based on V_(H). Residual V_(L) dimer of DNP-glycine was removedby gel filtration FPLC on a Superose 12 column in 100 mM potassiumphosphate, pH 6.8. Hybrid Fv reconstituted in this fashion has the sameaffinity for DNP-L-lysine as Fv purified from pepsin treated MOPC315 IgAthat was not reconstituted (data not shown). Because of convenience,myeloma-produced Fv(315) was used in this study. Protein purity wasassessed by electrophoresis through 14% polyacrylamide SDS gels (Laemmli(1970) Nature 227:680) followed by coomassie staining and by FPLC gelfiltration analysis using a Superose 12 column (flow rate of 0.5 ml/min,sample size of 100 micrograms).

The binding of ε-2,4-DNP-(L)-lysine (DNP Lys) by mutant wild type Fvproteins was assessed by titration of the intrinsic fluorescence ofMOPC315 Fv (Hochman et al. (1973) supra; Hochman et al. (1977) supra),as shown in FIG. 21. Wild type Fv (Fv(315)) and the Phe mutant(Fv(Y34F_(L))) bind DNP Lys with similar affinities at pH 6.8 (K_(D)=0.27±0.03 μM), while the His mutant (Fv(Y34H_(L))) binds this ligand6-fold less tightly (K_(D) =1.4±0.3 μM) (FIG. 4a) (values are reported±SE). These data suggest that the Tyr hydroxyl group is not importantfor recognition of the DNP moiety. DNP-aminoalkyl carboxylic acids ofdifferent lengths 6-9 (FIG. 16) were used to probe the location of thepositively charged imidazole ring with respect to the DNP site inFv(Y34H_(L)) at pH 6.0. The relative affinities of Fv(Y34H_(L)) forthese ligands (7>8>9>6) is different than that of the Fv(315 )(9>7>8>6). In general, Fv(Y34H_(L) binds 6-9 two-to 8-fold less tightlythan Fv(315), consistent with its lower affinity for DNP Lys at pH 6.8.In contrast to Fv(315), Fv(Y34H_(L)) binds 7 and 8 most tightly,suggesting a compensating charge interaction between thenegatively-charged carboxylate of the ligand and the protonatedimidazolium side chain.

The ability of wild type and mutant Fvs to hydrolyze esters 1-3 wasassayed by measuring the rate of coumarin release fluorimetrically at pH6.8 (Pollack et al. (1988) supra; Pollack (1989) supra). Fv(Y34H_(L))efficiently hydrolyzes 1 in a manner consistent with Michaelis-MentenKinetics (K_(m) =2.2±0.2 μM, k_(cat) =0.18±0.03 min⁻¹, k_(cat) /K_(m)=8.2×10⁴ M⁻¹ min⁻¹, pH 6.8) (FIG. 22, Table 2). Under these conditions,Fv(Y34H_(L)) turns over at least 11 times and still retains 44% of itsactivity. The loss of activity is likely due to the accumulation of theinhibitory reaction product 8. The Fv-catalyzed reaction wascompetitively inhibited with DNP-L-lysine (K_(i) =3.8±1.5 μM). Ester 2was a poor substrate (k_(cat) (2)=0.015 min⁻¹); and the hydrolysis of 3was not accelerated (k_(cat) (3)<0.005 min⁻¹). The fact that only thehydrolysis of 1 is significantly accelerated by Fv(Y34H_(L)) isconsistent with the preference for ligands 7 and 8, and the affinitylabelling data. The initial rate of hydrolysis of 1 by Fv(Y34H_(L)) atpH 6.8 is approximately 45-fold faster than the reaction with eitherFv(Y34H_(L)) or Fv(315) (Table 2). at pH 8.7, the reactivity ofFv(Y34H_(L)) and Fv(315) both increase, but differentially(v[Fv(Y34H_(L) ]/v[Fv(315)]=3.3, at 10 μM 1). Under these conditionsFv(Y34H_(L)) catalytically hydrolyzes 1 while Fv(315) reactsstoichiometrically. The increased reactivity of Fv(315) at higher pH maybe due to deprotonation of the tyrosine hydroxyl and subsequenttransesterification to form a relatively stable phenol ester.

                  TABLE 2                                                         ______________________________________                                        Substrate Specificity.sup.a,b of Fv(Y34F.sub.L)                                      Fv(Y34H.sub.L) Fv(315)  Fv(Y34F.sub.L)                                 ______________________________________                                        1      0.18 ± .03.sup.c                                                                          0.004    0.004                                          2      0.015          0.005    --                                             3      <0.005         --       --                                             ______________________________________                                         .sup.a Expressed as k.sub.cat, min.sup.-1                                     .sup.b Assay conditions as in FIG. 4b.                                        .sup.c SE (n = 3)                                                        

The imidazole acylating agent diethylpyrocarbonate rapidly andcompletely inhibits the hydrolysis of 1 at pH 6.0 (Pollack et al. (1989)supra; Holbrook (1973) Biochem. J. 131:729), supporting a catalytic rolefor His 34. The imidazole side chain of His 34 could catalyze thehydrolysis of ester by three mechanisms: i) nucleophilic attack on theester carbonyl to form a labile imidazolide intermediate which is thenrapidly hydrolyzed; ii) activation of an attacking water molecule; iii)electrostatic stabilization of the negatively-charged transition-statecreated by hydroxide ion attack on the ester carbonyl. The V_(max) ofFv(Y34H_(L)) hydrolysis of 1 increases linearly by only a factor ofthree from pH 5.6 to 8.6. This suggests that the rate limiting step ofhydrolysis does not involve simple hydroxide ion attack, since such areaction should be first order in hydroxide ion concentration. Combinedwith the fact that Fv(Y34H_(L)) is more active at higher pH (where theneutral form of imidazole predominates), it seems unlikely that theobserved rate acceleration is due simply to electrostatic stabilization.We presently have no evidence supporting either of the other mechanismsalthough imidazole derivatives hydrolyze active esters primarily by thenucleophilic pathway in aqueous solution.

In order to assess the contributions of the binding-site to hydrolysisof 1, the Fv(Y34H_(L)) catalyzed reaction was compared to the reactionwith 4-methylimidazole under the assay conditions (Pollack et al. (1989)supra). The ratio of the biomolecular rate constants (k_(cat)/K_(m))/k_(4-MeIm) is 9±3×10⁴. This rate acceleration is similar to thatof an antibody that contains an active site carboxylate which acts as aspecific base in the catalysis of an elimination reaction (Shokat (1989)Nature 338:269). The rate of hydrolysis of 1 by Fv(Y34H_(L)) can also becompared to that of MOPC315 Fab that has been chemically derivatizedwith an imidazole at Lys 52_(H) by a flexible tether 7 atoms in length(Example 2, above). Under similar conditions, the imidazole-derivatizedFab hydrolyzes 1 approximately 16 times less rapidly than Fv(Y34H_(L))(k_(cat) =0.011 min⁻¹, pH 7.0). The greater reactivity of Fv(Y34H_(L) )is likely due to the fewer number of degrees of freedom of the His 34side chain allowed by the protein backbone and surrounding side chains.In addition, the His imidazole may be better aligned for attack on theester carbonyl of 1, or for activation of a water molecule.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for preparing an antibody or antibodyfragment capable of promoting a chemical reaction involving theconversion of one or more reactants to one or more products, said methodcomprising:eliciting an antibody against a hapten which is a reactant, areactant analog, a transition state analog, or a multisubstrate analogand which further possesses structural and electronic features whichgenerate at least one preselected amino acid proximate the antibodycombining site, wherein the side chain of said preselected amino acid iscapable of promoting the reaction rate when the antibody or a fragmentthereof is exposed to the reactants.
 2. A method as in claim 1, whereinthe hapten comprises a polarized π system of a pyrimidine dimer in orderto elicit a tryptophan residue in the combining site of the antibody. 3.A method as in claim 1, wherein the hapten comprises a benzyl ammoniumion in order to elicit a carboxylate residue in the combining site ofthe antibody.